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Infection and Immunity, June 1999, p. 2867-2873, Vol. 67, No. 6
Unité de Génétique
Mycobactérienne,1 Laboratoire du
BCG,3 and Unité de
Physiopathologie de l'Infection,4 Institut
Pasteur, 75724 Paris Cedex, France, and Department of Medical
Microbiology and Immunology, Texas A&M University Health Science
Center, College Station, Texas2
Received 21 October 1998/Returned for modification 14 December
1998/Accepted 12 March 1999
New vaccines against tuberculosis are urgently required because of
the impressive incidence of this disease worldwide and the highly
variable protective efficacy of the current vaccine. The possibility of
creating new live vaccines by the rational attenuation of strains from
the Mycobacterium tuberculosis complex was investigated.
Two auxotrophic mutants of M. tuberculosis and M. bovis BCG were constructed by disruption of one of their purine biosynthetic genes. These mutants appeared unable to multiply in vitro
within mouse bone-marrow derived macrophages. They were also attenuated
in vivo in the mouse and guinea pig animal models. In guinea pigs, the
two mutants induced strong delayed-type hypersensitivity response to
purified protein derivative. In a preliminary experiment, the two
mutants were compared to the BCG vaccine for their protective efficacy
in a challenge against aerosolized virulent M. tuberculosis in the guinea pig model. Both mutants conferred some level of protection. These experiments demonstrate that the rational attenuation of M. tuberculosis could lead to the design of new
candidate live vaccines against tuberculosis.
Mycobacterium
tuberculosis, the etiological agent of tuberculosis (TB), remains
an important cause of human mortality and morbidity. Worldwide, 60 million people suffer from active TB and annually 7 million new cases
arise (10). The World Health Organization estimate is about
3 million deaths per year due to TB, of which more than 95% occur in
developing countries. In industrialized countries, the declining
incidence which had been observed since the beginning of the century
was reversed in the mid-1980s due in part to impaired social conditions
and the increasing frequency of AIDS (15).
Efficient chemotherapy exists but requires lengthy and expensive
treatments, making its widespread use and control difficult to achieve
in developing countries. Prophylactic vaccination against TB with the
attenuated strain of bovine mycobacteria, BCG (Mycobacterium bovis Bacillus Calmette-Guérin), is more cost-effective and
has indeed been used worldwide. Although BCG vaccination has provided protection against TB in certain populations, the variation in efficacy
of this vaccine in different field trials and its modest protective
effect against the adult form of the disease (estimated by
meta-analysis to be about 50%) (8) are points of major
concern. These considerations have led the World Health Organization to place TB control efforts, notably through the development of new vaccines, among its top priorities.
In the recent years, considerable efforts have been made toward the
identification of individual mycobacterial antigens involved in the
immune response to TB (25) with the aim of developing subunit vaccines. An alternative strategy to develop novel vaccines consists of constructing mutant strains of mycobacteria that are rationally attenuated. The recent development of genetic tools for
performing site-specific and random-site mutagenesis (4, 22)
in organisms from the M. tuberculosis complex now renders feasible the accomplishment of such a goal. Live vaccines should have
advantages over subunit vaccines in that (i) they represent a greater
pool of antigens which presumably should cover a wider range of T-cell
repertoires and (ii) they are generally more cost-effective to produce.
Moreover, attenuated mutants of M. tuberculosis should express homologous protective antigens which other mycobacterial strains lack and thus elicit a more specific and perhaps stronger protective immune response against virulence challenge. In support of
this hypothesis, the molecular analysis by Mahairas and collaborators (16) of genetic differences between M. bovis BCG
and its virulent counterparts M. bovis and M. tuberculosis clearly established the existence of regions of
deletion in the genome of BCG (representing about 30 kb in all), some
of which contain the open reading frames encoding the highly
immunogenic ESAT 6 and mpt64 antigens (the latter being absent from
certain BCG strains only [21]).
Among attenuated strains of intracellular bacterial pathogens, such as
salmonellae, yersiniae, brucellae, and corynebacteria (6, 9, 12,
18, 20, 23), auxotrophic mutants carrying defects in the
shikimate or the purine biosynthetic pathways were shown to be of
particular interest as potential live vaccines candidates because they
are attenuated in vivo and have the ability to retain their immunogenicity.
In mycobacteria, the only available attenuated auxotrophs are M. bovis BCG derivatives (11, 17). A BCG auxotroph
carrying a defect in the leucine biosynthetic pathway was unable to
grow in C57BL/6 mice (17) and to replicate inside human THP1
macrophages (2). This mutant, as well as other leucine,
methionine, and isoleucine-leucine-valine auxotrophs of BCG, were also
shown to be cleared from the organs of mice with severe combined
immunodeficiency disease (SCID mice); in contrast, the mice succumbed
to the wild-type BCG within 8 weeks. In addition, immunization with
these mutants provided BALB/c mice protection against a challenge with
M. tuberculosis (11). These results led the
authors to propose that the attenuated BCG strains could represent
potentially safe and useful vaccines against TB for populations at risk
for infection with human immunodeficiency virus. In M. tuberculosis, a leucine auxotroph was obtained by allelic exchange
(1), but unlike its BCG counterpart, it showed no
significant difference from the parental wild-type strain with respect
to growth in SCID mice, producing a fatal infection (3).
In a previous report, we described the isolation and characterization
of two genes from the M. tuberculosis purine biosynthetic pathway, purC and purL (13). We also
reported the construction by allelic exchange of a purC
mutant of M. tuberculosis (22). In this paper, we
describe the construction of a purC mutant of M. bovis BCG and provide evidence that both the M. tuberculosis and M. bovis purine auxotrophs are
affected in their survival inside mouse bone marrow macrophages and in
vivo in mice and guinea pigs. The delayed-type hypersensitivity (DTH)
responses to purified protein derivative (PPD) induced by both mutants
in a guinea pig model was investigated at 9 weeks postvaccination.
Finally, the level of protective immunity elicited by the two
auxotrophs against low-dose pulmonary challenge with virulent M. tuberculosis was evaluated in a guinea pig model of TB.
Bacterial strains and culture conditions.
Escherichia
coli XL1-Blue and the mycobacterial strains M. bovis
BCG strain Pasteur 1173P2, M. tuberculosis MT103 (isolated from a TB patient), and H37Rv (ATCC 27294) were cultured as previously described (22). When required, kanamycin (20 µg/ml),
gentamicin (10 µg/ml), or hypoxanthine (20 µg/ml), a purine
precursor, was added to the growth medium.
Plasmid construction.
Plasmid pMJ110 was constructed by
inserting the 3.7-kb BamHI fragment containing
purC::Km from pMJ101 into the BamHI
site of pJQ200 (22). This plasmid is unable to replicate in
mycobacteria. Plasmid pMJ111 was created by excising the 1,090-bp
EcoRV-HincII fragment from plasmid pMJ1,
harboring the purC gene flanked by 120 bp upstream and 80 bp
downstream, and cloning it into plasmid pOMK (13). pMJ111 is
able to replicate in mycobacteria.
Preparation and infection of mouse bone marrow macrophages.
Bone marrow cells were flushed from the femurs of 7- to 8-week-old
BALB/c mice (purchased from Iffa Credo, l'Arbresle, France) and
suspended in Dulbecco medium with low glucose (1 g/liter) and high
carbonate (3.7 g/liter) concentrations (Gibco BRL) and enriched with
10% heat-inactivated fetal calf serum (Dominique Dutscher), 10%
L-cell conditioned medium, and 2 mM glutamine. For the infection
assays, mouse bone marrow macrophages were seeded in eight-well Lab-Tek
chamber slides (Nalgene Nunc International) (5 × 104
cells/well) and infected with bacterial suspensions of either MT103,
BCG 1173P2, MYC1551, or MYC1552 at a multiplicity of infection of 1:1.
Cultures were incubated at 37°C in a 5% CO2 atmosphere. After 18 h, infection was terminated by removing the overlaying medium and washing each well three times with 500 µl of Hanks' buffered salt solution (Gibco BRL) before adding 400 µl of fresh culture medium per well. At days 1 (18 h), 4, 7, and 11, the number of
intracellular CFU was evaluated by plating appropriate dilutions of
lysed macrophages (in cell culture lysis reagent [Promega]) onto 7H10
or 7H10 supplemented with hypoxanthine. At each time point, a Lab-Tek
chamber slide containing infected macrophages and cultivated in the
same conditions was subjected to staining for acid-fast bacilli in
order to check macrophage viability. This infection experiment was
carried out in duplicate.
Mycobacterial multiplication in mice.
C57BL/6j or BALB/c
female mice (6 to 8 weeks old; purchased from CERJ, Le Genest St Isle,
France, or Iffa Credo) were infected intravenously either with
105 CFU of MYC1551 and MT103 or with 107 CFU of
MYC1552 and BCG in 0.5 ml of phosphate saline buffer. At every time
point, mice were euthanized with CO2, and the spleen, lung,
and liver were removed aseptically and homogenized either manually,
using two glass slides (for the spleen and lung), or in a Stomacher 80 (Seward) homogenizer (for the liver). Bacteria in the organs of
infected animals were enumerated by plating 10-fold serial dilutions of
organ homogenates onto 7H11 medium (supplemented with hypoxanthine for
the auxotroph mutants). The data were expressed as the geometric
means ± standard errors of the means (SEM) of counts obtained
with five to six mice.
Vaccination, challenge, and necropsy of guinea pigs.
Male
and female outbred Hartley strain guinea pigs (Charles River
Laboratories, Inc., Wilmington, Mass.) were used. Guinea pigs were
injected subcutaneously into the inguinal region with approximately
107 CFU of either wild-type BCG, the corresponding BCG
auxotrophic strain MYC1552, or the M. tuberculosis
auxotrophic mutant MYC1551 in a volume of 0.2 ml. Three, six, and nine
weeks postvaccination, three animals from each of the three vaccine
groups were euthanized. The number of viable mycobacteria carried by
one-half of the spleen and the right lower lobe of the lung was
determined by inoculating appropriate dilutions of organ homogenates
onto duplicate 7H10 plates supplemented with hypoxanthine. Nine weeks
postvaccination, all animals were infected via the respiratory route
with an infecting inoculum of viable H37Rv (empirically adjusted to
result in the inhalation of 5 to 10 viable organisms per animal) by use
of an aerosol chamber as previously described (24). Five
weeks after respiratory challenge, animals were euthanized, and the
spleen and right lower lobe of the lung were removed for bacterial culture.
Tuberculin skin test.
The DTH reaction was evaluated by the
intradermal injection of 0.1 ml of PPD containing 100 tuberculin units
(RT-23; Statens Seruminstitut) on a shaved area of the abdomen. The
mean diameter of induration was measured in millimeters and recorded
24 h later.
Statistical methods.
Analysis of variance was used to test
the effects of vaccination on tissue bacterial load. When significant
treatment effects were indicated, differences between means were
assessed by Duncan's multiple-range test. A 95% confidence level was
set for all tests. All analyses were performed with PC SAS 6.12 (SAS
Institute, Cary, N.C.).
Construction of purC mutants of M. bovis
BCG and M. tuberculosis.
An M. bovis BCG
purC mutant was obtained by using a classical allelic
exchange procedure consisting in the transformation of the wild-type
strain M. bovis BCG 1173P2 with a suicide vector carrying a
disrupted copy of the purC gene
(purC::Km) and selecting for kanamycin-resistant
(Kmr) transformants on 7H10 plates containing kanamycin and
hypoxanthine. M. bovis BCG 1173P2 was electroporated by
using 2 µg of plasmid pMJ110. Two Kmr transformants were
obtained. Their genomic DNAs were extracted and analyzed by Southern
blotting using the purC gene as a probe (Fig.
1). One of the two clones exhibited the
correct hybridization pattern for an allelic exchange mutant. The other
one corresponded to the wild type and was probably a Kmr
mutant of M. bovis BCG. The candidate clone was
phenotypically tested. As expected, it required purines or hypoxanthine
(a precursor of purine bases) to grow on 7H9 or 7H10 (Fig.
2). This mutant was renamed MYC1552.
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Persistence and Protective Efficacy of a
Mycobacterium tuberculosis Auxotroph Vaccine
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (47K):
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FIG. 1.
Southern blot analysis and hybridization profile of the
M. bovis BCG purC mutant. The hybridization
profiles of the DNA of Kmr transformants obtained in two
independent experiments are shown. Lane 1 corresponds to the M. bovis BCG purC mutant MYC1552; lanes 2 to 4 correspond
to transformants which have undergone a single crossover event at the
purC locus in another experiment; lane 5 is the wild-type
(WT) strain M. bovis BCG 1173P2. Chromosomal DNA were
digested with BamHI and probed for hybridization with the
purC gene (1,090-bp EcoRV-HincII
fragment from pMJ1).
View larger version (22K):
[in a new window]
FIG. 2.
Growth kinetics of MYC1552, MYC1551, MYC1551(pMJ111),
and their wild-type counterparts grown in 7H9 medium in the absence or
presence of the purine precursor hypoxanthine. The different strains
were grown in 7H9 medium (supplemented with hypoxanthine for MYC1551
and MYC1552) to an optical density at 600 nm (OD600) of
close to 1. These cultures were diluted in fresh medium in the absence
or presence of hypoxanthine (Hx) to reach an OD600 of 0.01 and then incubated at 37°C with 5% CO2. Growth of the
strains was monitored by measuring the OD600 of the
cultures every day.
Evaluation of the reversion frequency of MYC1551 and MYC1552.
To estimate the stability of the mutation introduced by allelic
exchange in M. bovis BCG and M. tuberculosis,
cultures of MYC1551 and MYC1552 grown in 7H9 containing kanamycin were
plated on 7H10 with kanamycin and hypoxanthine or 7H10 alone. In all the experiments (repeated independently four and two times for MYC1552
and MYC1551, respectively), no CFU was detected on 7H10 whereas 2 × 108 and 4 × 108 CFU in all were
obtained for MYC1551 and MYC1552, respectively, on 7H10 supplemented
with hypoxanthine and kanamycin. Therefore, the reversion frequency was
estimated to be lower than 10
8 events per cell and per
generation. The same experiment was repeated with cultures grown in 7H9
liquid medium without kanamycin, and the same results were obtained.
Growth characteristics of the purine auxotrophs in macrophages. The ability of the purine auxotrophs to persist and multiply within bone marrow macrophages from C57BL/6j mice was evaluated. Macrophages (5 × 104) were infected at a multiplicity of infection, of close to 1 with the parental and the mutant strains. Over a period of 15 days, the infection was followed by counting the viable bacteria (Fig. 3).
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Persistence of the auxotroph mutants and their parental counterparts in mice. M. tuberculosis (105 CFU) and M. bovis BCG (107 CFU) were used to infect BALB/c mice intravenously. The mice were killed at different time points, and viable bacteria in the liver, spleen, and lung were counted (Fig. 4 and 5).
For wild-type M. tuberculosis (Fig. 4), there was a large increase in the number of CFU in the three organs during the first 2 or 3 weeks: 2.5 logs in the spleen (day 14), 1 log in the liver (day 14), and 3 logs in the lung (day 21). At this time, the infection was controlled; the bacillary load was stabilized in both the lung and the spleen at 106 and 105 CFU per organ, respectively. In the liver, the bacterial burden decreased to reach a plateau at 104 CFU. For the M. tuberculosis purine auxotroph MYC1551, the situation was quite different. The initial multiplication was dramatically reduced, with less than 0.5-log increase in every organ at the peak occurring at day 7. The bacilli were then gradually eliminated from the three organs, becoming undetectable at day 63.
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DTH induced by the purine auxotrophs and protective efficacy in the guinea pig model. To evaluate the protective efficacy of the purine auxotrophs, the guinea pig was chosen because it is much more susceptible to M. tuberculosis infection than the mouse. Furthermore, BCG protection is easier to demonstrate in the guinea pig. While a maximum of 1 log of difference in the CFU number can be obtained in the lung and spleen of unimmunized compared to BCG-vaccinated mice, differences of 1 to 2 logs in the lung and 4 logs in the spleen are often obtained between naive and BCG-vaccinated guinea pigs following low-dose aerosol challenge (19).
Outbred Hartley strain guinea pigs were inoculated subcutaneously with 107 CFU of either BCG 1173P2, MYC1552, or MYC1551. Three animals from each treatment group were euthanized at 3, 6, and 9 weeks postinfection. The number of viable mycobacteria was determined in one half of the spleen and the right lower lobe of the lung. Three weeks postinfection, 870 ± 651 CFU of BCG 1173P2 was found in the spleens of three guinea pigs. In contrast, 90 CFU was recovered from only one of three guinea pigs infected with MYC1551, and no bacteria were found in any of the animals vaccinated with MYC1552. Six and nine weeks postinfection, no bacillus was recovered from the spleens of any animal of any of the three treatment groups. At no time point were bacilli recovered from the lungs of any vaccinated animal. These results show that the purine auxotrophs are attenuated even in the highly susceptible guinea pig as well as they were in the mouse model. The cutaneous DTH response to PPD was evaluated 9 weeks postvaccination. All of the vaccinated animals exhibited a detectable skin test reaction against tuberculin (Table 1). The mean induration diameters, measured 24 h following the intradermal injection, were 13.9 ± 0.9, 15.5 ± 1.0, and 11.1 ± 2.2, respectively, for the BCG-, MYC1552-, and MYC1551-infected guinea pigs.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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With the goal of developing a novel vaccine against TB, we constructed and evaluated the attenuation and protective efficacy of purC auxotroph mutant strains of M. tuberculosis and M. bovis BCG carrying a defect in the purine biosynthetic pathway. This approach was justified by the fact that attenuated strains are generally more potent than nonliving vaccines in stimulating cell-mediated immune responses which are effective against intracellular pathogens (7) and because, in theory, they produce most of the antigens normally expressed in vivo by the pathogens. Thus, immune responses are stimulated in ways which closely resemble those detected during normal infection. Moreover, by constructing an auxotrophic strain derived from M. tuberculosis, one would derive a vaccine candidate antigenically identical to the pathogen against which protection was desired.
As shown for other purine auxotrophs of intracellular pathogens which reside inside vacuoles (9, 18, 20), M. tuberculosis (MYC1551) and M. bovis BCG (MYC1552) purine-deficient mutants were attenuated both in an in vitro macrophage model and in vivo in mice and guinea pigs. Since the introduction of the purC gene on a plasmid into the M. tuberculosis auxotroph fully restores its ability to replicate and lyse the macrophages, one can conclude that restriction for growth inside these cells is due to the insertional disruption of the purC gene alone and not to polarity affecting the expression of adjacent genes.
In contrast to what was observed with their respective parental strains, the number of MYC1551 CFU in mouse bone marrow macrophages did not increase over a 15-day period of time but remained approximately constant, whereas the number of MYC1552 CFU steadily decreased from the first day postinfection. In the absence of purine bases in the medium, most likely resembling the situation within the phagosomal compartment, both auxotrophs were unable to multiply (Fig. 2). Thus, this difference in intracellular persistence between MYC1551 and MYC1552 probably reflects the different abilities of these M. tuberculosis and M. bovis BCG derivatives to resist phagocytic cells defenses and/or to make the phagosome in which they reside more hospitable.
The behavior of the auxotrophs in mice reflects the results obtained in isolated macrophages: the M. tuberculosis auxotroph MYC1551 was progressively eliminated after a lag period of 2 to 3 weeks during which the number of CFU in all organs remained almost constant, whereas elimination of the M. bovis BCG purC mutant MYC1552 began immediately after the infection. Overall, both strains were eliminated at about the same rate from mice. In guinea pigs, fewer MYC1551 than BCG and no MYC1552 were found in the spleens 3 weeks postimmunization. Finally, based on data obtained from the macrophage and guinea pig experiments in which the same infecting doses were used for all strains, it seems that both MYC1551 and MYC1552 are more attenuated than BCG.
The results of the protection studies, although preliminary because of the limited number of animals studied, indicated that all three strains (BCG, MYC1551, and MYC1552) had equal protective effects against an aerosol challenge with virulent M. tuberculosis H37Rv, as assessed by the reduction in CFU in the lungs of vaccinated guinea pigs. In the spleen, where more extensive protection is obtained, MYC1551 seemed to induce a protective effect compared to results for unimmunized controls, although it seemed slightly less efficient than that conferred by BCG. In the same organ, MYC1552 seemed to induce no protective response at all. The finding that MYC1552 shows a protective effect in the lungs but not in the spleen, unlike its M. tuberculosis counterpart and BCG, which show protective efficacy in both organs, could reflect the inability of the BCG auxotroph to control the dissemination of M. tuberculosis H37Rv following the aerosol infection and, thus, its inability to stimulate protective immunity with the same efficiency as the two other immunizing strains.
All three strains elicited strong DTH responses 9 weeks postinfection, suggesting that both auxotrophs are able to induce cell-mediated immune responses as does the BCG vaccine. Therefore, the differences in the protective efficacies of BCG, MYC1551, and MYC1552 may have resulted from differences in the quality of the cellular immune responses that they induce rather than in their ability to induce such responses. In fact, it is probable that an attenuated strain of mycobacteria needs to retain a limited ability to multiply in host cells and to disseminate and persist within the host in order to be able to induce protective immune responses. This idea was originally pointed out by Kanai's work (14), in which the protective efficacy of a streptomycin-dependent strain of M. tuberculosis was evaluated in guinea pigs and mice. In this system, streptomycin was used to induce the multiplication of the immunizing strain prior to challenge with virulent H37Rv tubercle bacilli. Experiments showed that only the M. tuberculosis that had multiplied during the immunization period conferred protection to the animals, with nonmultiplying bacteria displaying poor protection.
Thus, the way an attenuated strain establishes an infection in the host, probably more than persistence in itself, seems to be important for inducing protective immune responses. These observations highlight the difficulty inherent in using live vaccines to reach the right balance between attenuation and immunogenicity, since overattenuated bacteria may not produce in vivo some key antigens necessary for the induction of a protective immunity. The physiological state of the mutant bacilli might also influence the way their antigens are processed inside the macrophage and, thus, presented to the T lymphocytes.
Finally, this work provides evidence that rationally attenuated strains of M. tuberculosis can protect guinea pigs against pulmonary tuberculosis.
In further experiments with the MYC1551 and MYC1552 auxotrophs, it will be important to test whether immunizing the guinea pigs with higher doses of bacteria (108 CFU, for example) or with two or three doses of 107 CFU (injected 1 or 2 weeks apart) can improve the protective immune response that they induce. It will also be important to test whether MYC1551 and MYC1552 induce long-term protection by performing the aerosol challenge with virulent M. tuberculosis 4 or 6 months postimmunization. Also of great interest would be to evaluate the protective efficacy of the M. tuberculosis attenuated strains not only in the guinea pig model of tuberculosis but also in other models in which the BCG appears to be less potent and where improvements are thus required, such as models reflecting the case of individuals frequently exposed to environmental mycobacteria or developing TB from the reactivation of dormant bacilli.
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ACKNOWLEDGMENTS |
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This work was supported by the Institut Pasteur, by European Economic Community grant BMH4-CT97-2134, and by NIH grant AI 35207. M.J. is a recipient of a Fondation Mérieux grant.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institut Pasteur, Unité de Génétique Mycobactérienne, 25 rue du Dr. Roux, 75724 Paris cedex 15, France. Phone: 1 45 68 88 77. Fax: 1 45 68 88 43. E-mail: mjackson{at}pasteur.fr.
Editor: S. H. E. Kaufmann
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Materials and Methods
Results
Discussion
References
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