AgResearch, Wallaceville Animal Research Centre, PO Box 40063, Upper Hutt, New Zealand1
Author for correspondence: D. M. Collins. Tel: +64 4 922 1310. Fax: +64 4 922 1380. e-mail: desmond.collins{at}agresearch.co.nz
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ABSTRACT |
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Keywords: tuberculosis, Mycobacterium tuberculosis, vaccines, virulence, BCG, guinea pig
Abbreviations: PPD, purified protein derivative
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INTRODUCTION |
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In the last 10 years, an increased concern about tuberculosis together with many developments in immunology and molecular biology has aroused intense interest in producing better vaccines against the disease. There is general agreement that a successful vaccine will need to induce a type 1 immune response, but the detailed combination of responses that are required to induce optimum levels of protective immunity is not known (Snewin et al., 2000 ). This has made it difficult to specify which features of a vaccine are most important and therefore which particular type of vaccine is likely to prove most effective. A large range of possible types of both living and non-living vaccines are being actively researched (Collins, 2000
; Snewin et al., 2000
) and different types of vaccines may be useful for different purposes.
One approach is to modify the current vaccine and already a genetically modified form of BCG has been reported to produce better protection than BCG (Horwitz et al., 2000 ). The genetic causes of attenuation in BCG remained completely unknown until 6 years ago when it was shown that a block of nine genes, including the esat6 gene, was missing in BCG but was present in other strains of the M. tuberculosis complex (Mahairas et al., 1996
). More recently, specific inactivation of the esat6 locus was shown to cause partial, but not complete attenuation of an M. bovis strain (Wards et al., 2000
), and other blocks of genes that are absent (Behr et al., 1999
; Gordon et al., 1999
) or duplicated (Brosch et al., 2002
) in BCG have been identified. Add to this the likely presence of mutated genes which are yet to be identified and it is evident that considerable further work will be required to elucidate the role of the many genetic differences between BCG and clinical tuberculosis strains.
An alternative method of making a new live tuberculosis vaccine is to attenuate a virulent strain of the M. tuberculosis complex by introducing one or more easily identifiable mutations and several groups have shown that vaccine candidates can be produced in this way (de Lisle et al., 1999 ; Jackson et al., 1999
; Hondalus et al., 2000
). In the early stages of carrying out allelic exchange of ahpC in an M. bovis strain (Wilson et al., 1997
), using an approach that was eventually successful (Collins, 2001
), we had produced moderate numbers of illegitimate recombinants and investigated their potential use as tuberculosis vaccines. A total of 440 illegitimate recombinants were screened on minimal medium and four mutants were selected, all of which were found to be attenuated in guinea pigs. When guinea pigs inoculated with these mutants were challenged with virulent M. bovis, two of the mutants induced a level of protection that was in some respects better than that induced by BCG, although the differences were not significant (de Lisle et al., 1999
). In one of these mutants, a putative undecaprenol kinase gene was interrupted by illegitimate insertion of a DNA fragment that accompanied a 2 bp chromosomal deletion and, in the other, DNA fragment insertion was accompanied by a large DNA deletion of 15 kb containing 12 genes. None of the four mutants appeared to have any interrupted genes encoding enzymes for common metabolic pathways that are often associated with auxotrophy, such as amino acid, purine, pyrimidine or co-factor synthesis. Subsequently, we found these mutants were not true auxotrophs as 2-week-old cultures in complete medium were able to grow when inoculated into minimal medium. Re-examination of procedures revealed the mutants in the study had been subcultured into minimal medium from 5- to 10-week-old cultures in complete medium, at a stage when they would have been in stationary growth. Since the ability of strains of the M. tuberculosis complex to survive in the host in a stationary or lowered metabolic state is regarded as a crucial facet of tuberculosis pathogenesis (Colston & Cox, 1999
), we reasoned that it might be desirable to have a vaccine strain without this ability.
Regardless of this explanation, the use of 5- to 10-week-old cultures had produced two mutants with vaccine effectiveness at least equal to that of BCG, so a similar approach was used to produce mutants in this study. Eight recombinants were obtained by screening 1000 illegitimate mutants of M. bovis and these were produced by using either the original ahpC fragment or a second fragment made from another M. bovis gene. Identification of the mutated regions and virulence and vaccine testing of the strains was performed and two strains with vaccine properties at least as good as those of BCG were obtained.
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METHODS |
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Techniques for making recombinants and determining mutation sites.
The DNA fragment containing the interrupted ahpC gene (Fig. 1a) used for illegitimate recombination was prepared by NotI digestion as described previously (Wilson et al., 1997
). A DNA fragment containing the Rv3844 gene was also prepared. Briefly, a 2593 bp region of M. bovis (homologous to M. tuberculosis at position 1315715749 of Mtcy1A6; GenBank accession no. Z83864) was amplified using PCR primers containing PacI sites and cloned into pUHA9, a derivative of pBluescript KSII+ (Stratagene) which contains a PacI cloning site. A kanamycin resistance gene (neo) was inserted at the AatII site of Rv3844 by appropriate DNA digestion, blunt-end ligation and cloning. For electroporation, the plasmid containing this construct was digested with StuI and NotI which cut at positions equivalent to 13257 and 15209 respectively in the 1315715749 fragment of Mtcy1A6 to produce a 3·4 kb fragment (Fig. 1b
). The ahpC and Rv3844 plasmid digests were precipitated and washed in ethanol to remove electrolytes and electroporated into M. bovis using a high efficiency technique at 37 °C as described previously (Wards & Collins, 1996
). The electroporated cells were cultured on solid medium containing kanamycin and individual colonies were subcultured in liquid medium containing 20 µg kanamycin ml-1.
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Transcription of glnA2.
Exponential-phase cultures of M. bovis ATCC 35723 and WAg530 were harvested by centrifugation and RNA was extracted by cell disruption of 108 c.f.u. in the presence of 1 ml Trizol (Invitrogen) and 0·5 ml zirconium beads. After Trizol extraction, residual DNA was removed by incubation in DNaseI (Invitrogen) and RNA was further purified using an RNeasy Mini Kit (Qiagen). Reverse transcription was performed using gene-specific primers (designated RT-PCR in Table 1) and C. therm polymerase (Roche), according to the manufacturers instructions. PCR was performed in either a GeneAmp PCR System 9600 (Applied Biosystems) or a GeneAmp PCR System 9700 (Applied Biosystems) in 9600 emulation mode using the following programme: 1 cycle of 94 °C for 5 min; 32 cycles of 94 °C for 45 s, 60 °C for 30 s, 72 °C for 60 s; and 1 cycle of 72 °C for 7 min.
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Determination of vaccine efficacy.
For vaccination studies, groups of six DuncanHartley guinea pigs were vaccinated by subcutaneous injection in the right flank with 105 c.f.u. of one of the avirulent recombinant strains. A group of 12 animals was vaccinated with BCG and a control group of 12 animals was not vaccinated. Eight weeks post-vaccination, all animals were challenged with a single cell suspension of M. bovis WAg201 prepared by sonication for 30 s and filtration through an 8 µm membrane filter. Guinea pigs were infected via the respiratory route by using an aerosol chamber which produces droplet nuclei of the size appropriate for entry into alveolar spaces (McMurray et al., 1985 ; Wiegeshaus et al., 1970
). The concentration of viable M. bovis in the nebulizer fluid was empirically adjusted to result in the inhalation and retention of 210 viable organisms per guinea pig. The aerosolized solution contained approximately 4·8x104 c.f.u. ml-1. The requirement for this challenge dose had previously been estimated from the number of primary tubercles observed grossly in the lungs of non-vaccinated guinea pigs at 4 weeks post-infection. Five weeks after challenge, the animals were euthanased and autopsied, and body weight and gross pathology were recorded. Samples of spleen were subjected to mycobacterial culture and enumeration. Delayed type hypersensitivity to tuberculin injected intradermally was measured immediately before vaccination and prior to the animals being sacrificed using bovine PPD. Statistical analyses by ANOVA were performed on spleen weights and on log10 transformations of spleen and lung bacterial counts and numbers of macroscopic lesions in the spleen.
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RESULTS AND DISCUSSION |
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WAg526 and WAg529 both have large deletions at the sigG locus (Fig. 3b). WAg529 had greatly reduced virulence and WAg526 was avirulent. Since the deletion in WAg529 is nested within that of WAg526, it can be concluded that one or more of the extra genes of unknown function that were deleted in WAg526 (Rv0175Rv0178) contribute to loss of virulence as well as at least one of the genes absent in both recombinants. Of the two genes of known function identified as inactivated in both recombinants, sigG is a sigma factor gene belonging to the family which regulates extracytoplasmic functions and stress responses and bglS encodes a ß-glucosidase. Neither of these genes has been studied in detail but it has been reported that the expression of sigG is strongly down-regulated under conditions of low aeration (Manganelli et al., 1999
). The 5- to 10-week-old cultures used for screening would have had lowered levels of oxygen and the inability of these two strains to grow when transferred to fresh minimal medium as well as their considerable loss of virulence may indicate that sigG plays a significant role when the bacteria are returning from lower to higher levels of oxygen, a situation that may also occur in vivo.
WAg530 has a small deletion in the glnA2 gene encoding a putative glutamine synthetase and was found to be avirulent. Two other genes in the same chromosomal region (Fig. 3d), glnA2 and glnE, are also involved in glutamine synthesis. The glutamine synthetase encoded by glnA1, which has been extensively studied, is not actively exported from M. tuberculosis, but is found in the extracellular medium of M. tuberculosis cultures and may play a role in pathogenesis (Tullius et al., 2001
). The product of glnE is a regulator that by comparison to homologous enzymes in Streptomyces coelicolor controls glnA1, but not glnA2 (Parish & Stoker, 2000
). glnE has been shown to be essential (Parish et al., 2001
), but glnA2 has not been studied. In the annotation of DNA sequences from M. tuberculosis, glnA2 reads in the same direction as glnE and its coding sequence ends only 48 bp before the initiation codon of glnE. This region is identical in the M. bovis genome. This raised the possibility that these two genes may be in an operon and may be expressed co-ordinately. To test this possibility, we measured RNA production at various points in this region of the chromosome (Fig. 4
). RNA transcription of glnA2 upstream of the insertion site and of glnE occurred in both WAg530 and its parent strain and there was no transcript running from glnA2 to glnE in either strain. The only transcription difference between the strains was the absence in WAg530 of any glnA2 transcription downstream of the illegitimate insertion site in glnA2. These results indicate that glnA2 and glnE are not in an operon and are consistent with the earlier finding that glnE is an essential gene (Parish et al., 2001
). The loss of virulence in WAg530 is thus likely to be entirely due to disruption of glnA2 and not to downstream polar effects on glnE.
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Vaccination study
No delayed type hypersensitivity reactions were observed when the animals were tested prior to vaccination. All animals had positive skin test reactions immediately prior to sacrifice and there were no statistical differences between the responses of the groups of challenged animals, including the non-vaccinated group. Macroscopic lesions consistent with tuberculosis were observed in the lungs of the guinea pigs challenged with virulent M. bovis and the protection afforded by the vaccines against spread of infection was judged by the size of the spleens, the number of spleen lesions and the log(transformed c.f.u. of M. bovis) in the spleen and lung (Table 3). Cultures were not available for WAg530 and WAg531. There were a few liver lesions in most of the unvaccinated control animals, and in some animals vaccinated with WAg528, but they were not sufficient to make these groups significantly different from the other vaccinated groups that had no liver lesions. The five recombinants that were tested gave widely varying levels of protection against the spread of infection. WAg527 and WAg528 provided relatively poor protection, although the spleen c.f.u. were significantly different from controls. WAg531 gave intermediate protection. The best protection was provided by BCG, WAg526 and WAg530 and these could not be distinguished statistically (P<0·05). No spleen lesions were found for WAg526 and WAg530.
As in our earlier study (Wilson et al., 1997 ), several recombinants have produced results that, while not significantly different from those for BCG, do have some indices of protection that indicate a lower spread of infection than that achieved by BCG. In some recent vaccine models of tuberculosis in which animals were first exposed to environmental mycobacteria, BCG provided little or no protection against tuberculosis (Brandt et al., 2002
; Buddle et al., 2002
), although in the second of those studies chemical mutants of M. bovis gave significant protection. These differences in protection may result from differences in persistence between BCG and other avirulent strains or from stimulation of a qualitatively different immune response. Whether the strains developed in this study would give significantly better protection than BCG if one of these more recent vaccine models was used or if the vaccination protocol was altered in some other way to exemplify the differences between protective vaccines requires further experimentation. The two recombinants that gave poorer protection (WAg527 and WAg528) are both strains that lack an active pckA gene. WAg531, which also lacks an active pckA gene, provided protection that was not significantly different to that provided by BCG, although it did allow a higher mean spleen weight and lesion count than BCG, WAg526 and WAg530. These results show that while inactivating pckA does create avirulent strains of M. bovis, those strains do not appear to have the potential to provide a better vaccine than BCG. More generally, it also shows that avirulent strains of the M. tuberculosis complex are not all able to stimulate a good level of protection against tuberculosis even when their inoculation into guinea pigs results in normal skin test reactivity.
The results described here support the approach used in this and the previous study (Wilson et al., 1997 ; de Lisle et al., 1999
) of screening stationary-phase cultures for their ability to grow in minimal medium and testing the virulence and vaccine potential of strains identified by this selection. Together with our earlier study, a total of 12 recombinants (0·8%) have been isolated from 1440 tested of which nine (0·6%) are avirulent. None of the genes inactivated in these nine avirulent strains has been reported in other studies as being important for virulence of strains of the M. tuberculosis complex. Several of the recombinants provide protection against the spread of tuberculosis that, while not significantly different to that provided by BCG, results in slightly better indices of protection than BCG. Whether the recombinants produced here would provide better protection against tuberculosis than BCG if they were tested under more stringent conditions remains to be determined, but the current results encourage belief that a better live vaccine than BCG may be attainable.
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ACKNOWLEDGEMENTS |
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Received 26 March 2002;
revised 5 July 2002;
accepted 8 July 2002.