Attenuated strains of mycobacteria

Attenuated strains of Mycobacterium, particularly species of the tuberculosis complex, have the mycobacterial cell entry (mce) gene functionally disabled. The gene may be disabled by an insertion into the gene which disrupts the mycobacterial cell entry function thereof of a selectable marker which is used for screen for homologous recombinants in which a double cross-over event has been effected. The attenuated strains may be used in the immunization of hosts against Mycobacterium disease.

FIELD OF INVENTION 
The present invention relates to the field of molecular immunology and, in 
particular, to attenuated strains of Mycobacterium and immunogenic 
preparations comprising the same. 
BACKGROUND TO THE INVENTION 
Tuberculosis (TB) is a major cause of mortality throughout the world, 
particularly in developing countries. There are about 8 to 9 million new 
cases of clinical disease reported every year and the number of deaths is 
estimated to be about 3 million. In the U.S. the trend of steady decline 
in TB has reversed and the problem is compounded by increasing numbers of 
drug-resistant strains. The tuberculosis complex is a group of four 
mycobacterial species that are genetically closely related. The three most 
important members are Mycobacterium tuberculosis, the major cause of human 
TB; Mycobacterium africanum, a major human pathogen in some populations; 
and Mycobacterium bovis, the cause of bovine TB. None of these 
mycobacteria is restricted in being pathogenic for a single host species. 
In addition to being an important human disease, TB is also a major 
veterinary problem in many countries. Infection of cattle with M. bovis 
results in bovine TB and all animals showing any signs of infection are 
systematically slaughtered. The economic losses are thus extensive, and 
furthermore, cattle can serve as a reservoir for human disease. 
In a majority of cases of infection, inhaled tubercle bacilli are ingested 
by phagocytic alveolar macrophages and are either killed or grow 
intracellularly to a limited extent in local lesions called tubercules. In 
this way the infection is limited and the primary sites of infection are 
walled off without any symptoms of disease being observed. Such 
individuals have a lifetime risk of about 10% for developing active 
disease. In a latter eventuality, bacilli spread from the site of 
infection in the lung, through the lung and via lymphatics or blood to 
other parts of the body producing characteristic solid caseous 
(cheese-like) necrosis in which bacilli survive. If the necrotic reaction 
expands breaking into a bronchus, or in the worst case, if the solid 
necrosis liquefy, a rapid proliferation of the bacilli occurs. The 
pathological and inflammatory processes set in motion then produce the 
characteristic weakness, fever, chest pain, cough and bloody sputum which 
are the hallmarks of active TB. 
Effective treatment of TB with antibiotics exists. However, this is 
expensive and requires prolonged administration of a combination of drugs. 
There is a problem in compliance with the drug administration regime 
because of the extended time periods involved and this has contributed to 
the appearance of drug-resistant strains. There is a recognized vaccine 
for TB which is an attenuated form of M. bovis, known as BCG (bacille 
Calmette Guerin). This strain was developed in 1921 and the basis for its 
attenuation is still not known (ref. 1--throughout this application, 
various references are cited in parentheses to describe more fully the 
state of the art to which this invention pertains. Full bibliographic 
information for each citation is found at the end of the specification, 
immediately preceding the claims. The disclosure of these references are 
hereby incorporated by reference into the present disclosure). The 
efficacy of BCG as a TB vaccine is a subject of controversy and has been 
estimated in various trials to be anywhere between 0 and 70%. 
The molecular basis for the virulence and pathogenesis of M. tuberculosis 
has not been extensively described. Some virulence factors, particularly 
those related to the sigma factors have been recently identified (ref. 2). 
M. tuberculosis can enter non-phagocytic cells in culture, such as HeLa 
cells (ref. 3) and once inside can multiply and survive. Recently, a 
protein encoded by a DNA fragment (1535 bp long) from a strain of M. 
tuberculosis (H37Ra) was reported to mediate the entry of the bacterium 
and its survival in mammalian cells (ref. 4). This DNA fragment when 
introduced into a non-pathogenic strain of E. coli is able to confer 
invasiveness to the bacterium, and survival for up to 24 hours in human 
macrophages. The mce (mycobacterial cell entry) gene was mapped to an Open 
Reading Frame (ORF) extending from position 182 to 810 on the 1535 bp DNA 
fragment mentioned above and encodes a protein of molecular weight between 
22 and 27 kDa. Subsequent work has shown the gene described in ref. 4 is 
not a full length gene. 
In copending U.S. patent application Ser. No. 08/677,970 filed Jul. 10, 
1996, assigned to the assignee hereof and the disclosure of which is 
incorporated herein by reference, there is described the isolation and 
characterization of genes encoding proteins of mycobacteria associated 
with cell binding and cell entry and the protein encoded thereby. This 
gene is referred to herein as the Mycobacterial cell entry (mce) gene and 
the encoded protein the Mycobacterial cell entry protein (Mcep). 
Mycobacterial infection may lead to serious disease. It would be 
advantageous to provide attenuated strains of Mycobacterium wherein the 
mycobacterial cell entry gene is disabled, and immunogenic preparations 
including vaccines comprising the same. 
SUMMARY OF INVENTION 
The present invention provides attenuated strains of Mycobacteria which are 
useful in immunogenic compositions. In accordance with one aspect of the 
present invention, there is provided an attenuated strain of Mycobacterium 
wherein the mycobacterial cell entry (mce) gene is functionally disabled. 
By functionally disabling the mce gene, the ability of the Mycobacterium 
to invade and infect cells is removed. This attenuation permits the novel 
strains provided herein to be used in immunogenic compositions for 
administration to a host to generate an immune response. 
The mce gene may be functionally disabled by an insertion into the gene 
such as to disrupt the mycobacterial cell entry function thereof. The mce 
gene also may be functionally disabled by deleting at least a part of the 
gene from the wild-type strain. In addition, mutagenesis of the mce gene 
may be used to attenuate the wild-type strain. 
The mutant strain of Mycobacterium may be prepared by any convenient 
procedure. Homologous recombination conveniently may be used to replace 
the mce gene of the wild-type strain of Mycobacterium by a double 
cross-over event with a disabled mce gene. 
The present invention is broadly applicable to strains of Mycobacterium, 
particularly a species of the tuberculosis complex, including M. 
tuberculosis and M. bovis. 
In another aspect of the invention, there is provided a method of forming 
an attenuated strain of Mycobacterium, which comprises effecting allelic 
exchange of a mutant mycobacterial cell entry (mce) gene which is 
functionally disabled for a mycobacterial cell entry gene in a wild-type 
strain of Mycobacterium. 
The mutant mce gene may contain a selectable marker, so that the attenuated 
strain of mycobacterium formed in the allelic exchange may be detected on 
the basis of the presence of the selectable marker therein. 
A further aspect of the invention provides an immunogenic composition 
comprising the attenuated strain provided herein. Such immunogenic 
composition may be formulated as a vaccine for in vivo administration to a 
host to confer protection against disease caused by a virulent strain of 
Mycobacterium. The host may be a primate including a human. 
The present invention includes, in a further aspect thereof, a method of 
generating an immune response in a host comprising administering thereto 
an immunoeffective amount of the immunogenic composition provided herein. 
A yet further aspect of the invention provides a method of producing a 
vaccine for protection against a disease caused by infection by a virulent 
strain of Mycobacterium, which comprises administering the immunogenic 
composition provided herein to a first host to determine an amount and 
frequency of administration thereof to confer protection against the 
disease; and formulating the immunogenic composition in a form suitable 
for administration to a treated host in accordance with the determined 
amount and frequency of administration. The treated host may be a human. 
The attenuated strains of Mycobacterium provided herein are useful as a 
live vaccine against diseases caused by Mycobacteria. Advantages of the 
present invention include the provision of safer and attenuated strains of 
Mycobacterium for the preparation of immunogenic compositions, including 
vaccines, and for the generation of immunological and diagnostic reagents.

GENERAL DESCRIPTION OF THE INVENTION 
The use of BCG herein is a useful means of illustrating the broader 
application of the present invention to functionally disabling the 
mycobacterial cell entry gene in a strain of Mycobacterium, including any 
of the species of the tuberculosis complex, including Mycobacterium 
tuberculosis. The provision of the strain of Mycobacterium in which the 
mce gene is functionally disabled provides attenuated strains of 
Mycobacterium which may be used safely in immunogenic compositions. 
Referring to FIG. 1, there is illustrated therein the construction of a 
disrupted mce gene. Plasmid pBCGcepX, the preparation of which is 
described in the above mentioned U.S. application Ser. No. 08/677,970 and 
deposited under ATCC No. 97511, is digested with restriction enzyme BsiWI 
to cut the mce gene at the restriction site. In FIG. 1 only the 4.7 kb 
XhoI fragment of the plasmid is shown. 
The hygromycin resistance gene (hyg) of Streptomyces hygroscopices is 
isolated from a plasmid pIDV6, obtained from ID Vaccines, by digestion 
with restriction enzyme NotI. Following separation of a 2.5 to 3 kb 
fragment, restriction enzyme BspHI is used to isolate a 1.3 kb fragment 
containing hyg gene. 
The hyg gene is ligated with the BsiWI digested plasmid pBCGcepX and the 
ligate used to transform E. coli. Following selection for hygromycin 
resistance, transformants are grown and the plasmid isolated. Plasmid 
pBCGcepX-H, produced by this procedure, has the hyg gene inserted into the 
mce gene, in the opposite direction. 
The plasmid BCGcepX-H is linearized and the linearized plasmid is used to 
transform a Mycobacterium strain, for example, M. bovis BCG, by homologous 
recombination. The construction by homologous recombination of mutants 
deficient in some metabolic genes has been achieved recently in slow 
growing mycobacteria (refs. 5, 6, 7). The suppression of key metabolic 
enzymes was expected to lead to the generation of less virulent strains, 
with little success so far (ref. 8). 
Screening of recombinant events may be performed by PCR analysis. 
Hygromycin resistant M. bovis BCG colonies are subjected to PCR analysis 
using a pair of primers corresponding to appropriate portions of the mce 
gene. As seen in FIG. 1, primer P4414 (SEQ ID NO: 1) and P4448 (SEQ ID 
NO:2) (the nucleic acid sequences of the primers are shown in Table 1 
below), are used for PCR amplification. Such primers generate a 572 bp PCR 
product for a wild-type strain while integration of the mutant mce gene by 
homologous recombination with double cross-over yields a 1.9 kb product. 
For a random DNA integrate or a single cross-over, two fragments are 
amplified. 
Three mutants (BCG-65, BCG-73, BCG-83) produced only a 1.9 kD PCR-amplified 
fragment, consistent with homologous recombination causing replacement of 
native mce gene by a disrupted copy of the gene. FIG. 2 shows the results 
of the PCR analysis. The wild-type strain produced a 572 bp fragment while 
a single cross-over mutant produced both fragments. 
In order to further assess the recombinant BCG as to the proper integration 
of the functionally-disabled mce gene, a Southern blot was performed. This 
required isolating the chromosomal DNA from the recombinant BCG colonies 
and digesting them with restriction endonucleases, and transferring the 
DNA fragments separated on the agarose gel to a nylon membrane. The probe 
for the mce gene was PCR amplified from M. tuberculosis H37RV DNA as 
described in Example 6 below. The 1.6 kb probe was used to verify the 
double cross-over events that occurred in BCG-65, BCG-73 and BCG-83. These 
strains represent attenuated BCG containing the functionally disrupted mce 
gene. 
To show that these attenuated BCG no longer produce the cell entry protein, 
Western blots were performed on cell lysates produced by sonication of the 
cells. A mycobacterial strain with a disrupted gene would not be able to 
make the Mce protein and, therefore, a mouse monoclonal antibody to the 
mycobacterial cell entry protein would not recognize any protein from this 
strain, as described in Example 7 below. FIG. 4, lane 1, clearly shows 
that such attenuated BCG, BCG-65, does not make any mycobacterial cell 
entry protein. A single cross-over or non-homologous recombinant, BCG-69, 
was not disrupted in the mce gene and still produced the wild-type 
mycobacterial cell entry protein (FIG. 4, lane 2). 
Biological Deposits 
A vector that contains the gene encoding a mycobacterial cell entry protein 
and having a molecular weight of between about 45,000 and about 60,000 
from the M. bovis strain BCG that is described and referred to herein has 
been deposited with the American Type Culture Collection (ATCC) located at 
10801 University Boulevard, Manassas, Va. 20110-2209, USA, pursuant to the 
Budapest Treaty and prior to the filing of this application in connection 
with application Ser. No. 08/677,970 referred to above. Samples of the 
deposited vectors will become available to the public upon grant of a 
patent based upon this or the aforementioned United States patent 
application and all restrictions on access to the deposit will be removed 
at that time. Viable samples will be provided if the depository is unable 
to dispense the same. The invention described and claimed herein is not to 
be limited in scope by the biological material deposited, since the 
deposited embodiment is intended only as an illustration of the invention. 
Any equivalent or similar vectors that encode similar or equivalent 
antigens as described in this application are within the scope of the 
invention. 
Deposit Summary 
______________________________________ 
ATCC 
Deposit Designation 
Date Deposited 
______________________________________ 
Plasmid pBCGcepX 
97511 April 11, 1996 
______________________________________ 
EXAMPLES 
The above disclosure generally describes the present invention. A more 
complete understanding can be obtained by reference to the following 
specific Examples. These Examples are described solely for purposes of 
illustration and are not intended to limit the scope of the invention. 
Changes in form and substitution of equivalents are contemplated as 
circumstances may suggest or render expedient. Although specific terms 
have been employed herein, such terms are intended in a descriptive sense 
and not for purposes of limitation. 
Example 1 
This Example illustrates the recombinant DNA methods used herein. 
Restriction enzymes and cloning vectors were obtained from several sources 
including New England Biolabs, Life Technologies, Boehringer Mannheim and 
Stratagene. The enzymes and buffers for the PCR were purchased from 
Perkin-Elmer or Sangon Corporation and used as per the manufacturers 
protocols. 
Reagents used in DNA isolation protocols were purchased from Sigma 
Biochemicals. Most recombinant DNA manipulations were performed using 
standard protocols (ref. 10). Sequences of double stranded plasmid DNA 
were determined using the Taq Dye Deoxy Terminator cycle sequencing kit 
(Applied Biosystems) on a GeneAmp PCR system 9600 (Perkin-Elmer) and a run 
on a DNA analysis system, model 370A (Applied Biosystems). The sequence 
was assembled and processed using the IG software (IntelliGenetics Inc). 
The synthesis of oligonucleotides used as primers was performed using an 
Applied Biosystems (380B) synthetizer. The synthetic oligonucleotides were 
purified on OPC cartridges supplied by Applied Biosystems according to the 
manufacturers protocol. 
Example 2 
This Example illustrates construction of the disrupted mce gene. 
5 .mu.g of plasmid pBCGcepX (ATCC #97511) were digested with restriction 
enzyme BsiWI (NEB Biolabs) for 2 hours at 37.degree. C. in 25 .mu.l final 
volume. 3 .mu.l of Nick translation buffer, 1 .mu.l of dNTP's (2 mM) and 2 
units of Klenow DNA Polymerase (Boehringer Mannheim) were added to the 
solution and it was incubated for 30 min at room temperature. 120 .mu.l of 
water were added and a phenol-chloroform extraction was performed by 
mixing: 75 .mu.l of phenol (Life Technologies) and 75 .mu.l of 
chloroform-isoamyl acid (24:1, v:v) to the solution. The tube was spun 
(12000.times.g for 2 min) and the aqueous phase was transferred to a fresh 
tube. 300 .mu.l of ice-cold 100% ethanol was added, the DNA was pelleted 
by centrifugation (12000 g for 15 min at 4.degree. C.), and washed with 1 
ml of 70% ethanol. The DNA was air dried at room temperature and 
resuspended in 40 .mu.l of water. 3 units of Calf Intestinal Alkaline 
Phosphatase (Boehringer Mannheim) were added and the mixture was incubated 
at 37.degree. C. for 1 hour in 50 .mu.l volume final. The DNA was purified 
from an agarose gel, and resuspended in 30 .mu.l of water. 
To isolate the hygromycin resistance gene (hyg) of Streptomyces 
hygroscopicus, 18 .mu.g of plasmid pIDV6 (obtained from Dr Horwitz, 
University of California, Los Angeles, Calif.) were digested with the 
restriction enzyme NotI (NEB Biolabs) for 3 hours at 37.degree. C. in 60 
.mu.l volume final. The digestion of plasmid pIDV6 with NotI resulted in 
two products, namely a 2.5 to 3 kb fragment containing the hyg gene and a 
larger fragment. The 2.5 kb band was purified and resuspended in 20 .mu.l 
of water. The restriction enzyme BspHI (NEB Biolabs) was added to the DNA 
and the mixture was incubated at 37.degree. C. for 2 hours 30 min, in 30 
.mu.l final volume. 3.5 .mu.l of Nick translation buffer, 1 .mu.l of 
dNTP's (2 mM) and 2 units of Klenow DNA Polymerase (Bochringer Mannheim) 
were added to the solution and the mixture was incubated for 30 min at 
room temperature. The digest was run on a 0.8% agarose gel, and consisted 
in two products, namely a 1.3 kb fragment and a smaller one. The larger 
piece of DNA, containing the hyg gene, was purified from the gel and 
resuspended in 15 .mu.l of water. 
The ligation was performed in a final volume of 20 .mu.l, using 1 .mu.l of 
plasmid pBCGcepX digested by BsiWI and treated as described above and 4 
.mu.l of the hyg gene isolated as described above. 1.5 units of T4 DNA 
Ligase (Life Technologies) were used in this reaction. The mixture was 
incubated overnight at 16.degree. C. to ligate the hyg gene with the 
digested pBCGcepX plasmid. 
2 .mu.l of the ligation mixture were used to transform 70 .mu.l of 
electro-competent E. coli HB101 cells, and 100 .mu.l of the transformation 
solution were plated onto Luria-Bertani agar (LB agar), with 100 .mu.g/ml 
of ampicillin and 200 .mu.g/ml of hygromycin B (Boehringer Mannheim). A 
few transformants were isolated and grown up. The plasmids were isolated 
using a kit for high grade plasmid purification (Qiagen) and sequenced. 
One of them, plasmid pBCGcepX-H, had the hyg gene inserted in the mce 
gene, in the opposite direction (see FIG. 1). 50 .mu.g of plasmid 
pBCGcepX-H were digested with the restriction enzyme Apal (Life 
Technologies) for 3 hours at 30.degree. C. in 200 .mu.l final volume. 
After incubation, 100 .mu.l of water were added and the DNA was purified 
by phenol extraction, followed by two phenol-chloroform extractions. The 
aqueous solution was transferred to a new tube, 35 .mu.l of 3M sodium 
acetate were added, the DNA was precipitated by adding 1 ml of ice-cold 
100% ethanol. The DNA was pelleted by centrifugation (12000 g for 10 min 
at 4.degree. C.), washed with 70% ethanol, air dried and resuspended in 25 
.mu.l of water. The concentration of DNA was determined by reading the OD 
at 260 nm. 
Example 3 
This Example illustrates transformation of M. bovis BCG with plasmid 
pBCGcepX-H. 
Electrocompetent M. bovis BCG cells were prepared using a modification of a 
protocol already described (ref. 9). 500 .mu.l of a frozen stock of 
Connaught M. bovis BCG strain were used to inoculate 10 ml of 7H9-ADC-Tw 
broth and incubated with shaking at 37.degree. C. for three days. Two ml 
of this preculture were used to inoculate 100 ml of 7H9-ADC-Tw broth and 
incubated at 37.degree. C. with shaking for three days. 1.5 g of glycine 
(Boehringer Mannheim) diluted in 10 ml of water and sterile-filtered was 
added to the culture and the culture was incubated one more day. 
The electrocompetent cells were spun down (4000 g for 15 min) and 
sequentially washed in 100, 50, 25, 10 ml of 10% glycerol. The cells were 
eventually resuspended in 3 ml of 10% glycerol. 
A 0.25 ml aliquot of resuspended cells was mixed with 3 .mu.g of linearized 
plasmid pBCGcepX-H, the mixture was incubated on ice for 10 min and 
subjected to electroporation in a 0.2 cm cuvette using a BioRad apparatus 
(BioRad,) at a setting of 2.5 kV, capacitance of 25 .mu.F and pulse 
controller to 1000.OMEGA.. The cells were then placed on ice for 10 min, 
resuspended in 1 ml M-ADC-TW broth and incubated for 3 hours with shaking 
at 37.degree. C. The transformed cultures were spread on 7H10 agar plates 
containing 50 .mu.g/ml of hygromycin B and 100 .mu.g/ml of cycloheximide 
(Sigma) and incubated at 37.degree. C. for 3 to 4 weeks. 
Example 4 
This Example illustrates PCR amplification of the M. bovis BCG colonies. 
Screening of recombinant events was performed by PCR reactions. 
Hygromycin-resistant M. bovis BCG colonies, prepared as described in 
Example 3, were isolated, used to inoculate 3 ml of 7H9-ADC-Tw broth, and 
incubated for three days at 37.degree. C. 1 ml of this culture was 
transferred to a microfuge tube, and spun down (12000 g for 10 min) to 
pellet the cells. The cells were resuspended in 50 .mu.l of water, boiled 
for 10 min and immediately placed on ice. The amplification reactions were 
carried out using the "Hot Start" procedure. Essentially, a 40 .mu.l 
reaction mix containing dNTP's (0.2 mM in 100 .mu.l final volume), buffer 
and a pair of primers (P4414, SEQ ID NO:1, and P4448, SEQ ID NO:2; 100 pM 
of each, see Table 1 below for identification of the primers) was prepared 
in thin-wall Eppendorf tubes. To each tube, a bead of wax (PCRGem 100, 
Perkin-Elmer) was added and the tube was heated to 70.degree. C. for 5 
min. Subsequently, the tube was cooled at room temperature for 5 min and a 
reaction mix (60 .mu.l) containing buffer, 1 unit of enzyme and 25 .mu.l 
of the colony preparation was added. The tubes were then placed in a 
Perkin-Elmer Cetus thermal cycler and a cycling sequence started based on 
the following parameters: 
Step 1: 2 min at 99.degree. C.; 
Step 2: 45 sec at 98.degree. C.; 45 sec at 60.degree. C.; 1 min 30 sec at 
72.degree. C.; repeated for 25 cycles; 
Step 3: 10 min at 72.degree. C.; 
Step 4: maintain at 4.degree. C. 
The tubes were stored at 4.degree. C.; aliquots of 10 .mu.l were run on a 
0.8% agarose gel and the electrophoretic patterns visualized and 
photographed. 
The set of primers used generated a 572 bp PCR product for wild type BCG 
strain, while integration by homologous recombination with double 
cross-over yielded a 1.9 kb product. If the DNA integrated randomly or by 
a single cross-over, then two fragments were amplified. Analysis of 88 
transformants by PCR analysis showed three mutants (BCG-65, -73, -83) 
yielding only a 1.9 kb fragment, as expected from homologous recombination 
causing replacement of the native mce gene by a disrupted copy of the 
gene. The 1.9 kb and 572 bp fragments were amplified for all the other 
transformants. 
FIG. 2 shows the results of the PCR screening described above. As may be 
seen therein, the mutant strain wherein a double cross-over event has 
caused replacement of the native mce gene by a disrupted form of the gene 
contained a 1.9 kb fragement (lanes 65, 73). The wild-type strain 
contained the 572 bp fragment (lane wt) while a random-integrate or a 
single cross-over mutant contained both the 572 bp fragment and the 1.9 kb 
fragment (lane 69). 
Example 5 
This Example illustrates the preparation of genomic DNA from M. bovis BCG. 
Genomic DNA from BCG cultures was extracted using a modification of a 
technique already described (ref. 11). 50 ml of a 14 days BCG culture was 
centrifuged (6000 g for 10 min) to pellet the cells. The pellet was 
incubated for one hour at 37.degree. C. in 1 ml of TE buffer (10 mM 
Tris-HCl, pH7.5 and 1 mM EDTA) containing 200 .mu.g/ml of proteinase K 
(Life Technologies) and 10 .mu.g/ml of hen egg-white lysozyme (Sigma, St 
Louis, Mo., USA). After centrifugation (12000 g for 5 min), the pellet was 
resuspended in 1 ml of DNAzol (Life Technologies), transferred to a 2 ml 
screw-capped tube filled to a quarter with glass beads (106 .mu.m or 
finer, Sigma) and vortexed vigourously for 10 min. The beads were allowed 
to settle and the supernatant was transferred to a fresh tube and 
centrifuged for 10 min at room temperature. The resultant lysate was 
transferred to a new tube and the DNA was precipitated by adding 0.5 ml of 
100% ethanol. The tube was inverted several times to mix the materials and 
the mixture was incubated at room temperature for 3 to 5 min. The tube was 
spun (at 1000 g for 2 min) to pellet the DNA, the supernatant discarded, 
the pellet washed twice with 1 ml of 95% ethanol, air-dried at room 
temperature and resuspended in 200 .mu.l of TE buffer. The quantity of DNA 
was estimated by measuring the optical density (OD) at 260 nm in a 
spectrophotometer. This protocol yielded approximately 80 .mu.g of DNA. 
Example 6 
This Example illustrates the preparation of the DIG-labelled mce probe and 
Southern hybridization of BCG DNA digests. 
PCR reactions were carried out on 500 ng of M. tuberculosis H37Rv DNA, 
using primers P4973 (SEQ ID NO:3) and P4974 (SEQ ID NO:4), located at the 
extremities of the ice gene of M. bovis BCG. PCR reactions were carried 
out as described in Example 4, except that the template was 500 ng of M. 
tuberculosis H37Rv DNA instead of 25 .mu.l of a colony DNA preparation. 
The amplification product (1.6 kb) was isolated by excising the band from 
a 0.8% agarose gel and extracting the DNA. The isolated DNA was labelled 
with DIG-dUTP, using the DIG-labelling kit (Boehringer-Mannheim), 
following the supplier's instructions. This procedure yielded the probe 
identified herein as PMCE. The sequence of the mce gene of M. tuberculosis 
H37Rv is 99% identical to the mce gene of M. bovis BCG. 
8 .mu.g of BCG DNA was digested in a 40 .mu.l final volume, for 3 hours at 
37.degree. C., with SacI or XhoI restrictions enzymes. The digests were 
run out on a 0.8% agarose gel. The gel was transferred to a nylon membrane 
(GeneScreen Plus, Dupont) using standard reagents and protocols and the 
DNA fixed to the membrane. 
The membrane was prehybridized, hybridized overnight at 65.degree. C. with 
the labelled probe PMCE and subsequently washed. The membrane was 
processed following the instructions of the kit supplier (Boehringer 
Mannheim). The blot was exposed to a film for 3 min at room temperature 
and the radiograph developed (see FIG. 3). 
FIG. 3 shows the results of the Southern Blot analysis performed as 
described above. Lanes 1 to 3 show the results for the SacI digests. The 
wild-type strain (Lane 1) gave a single band as 5.2 kb while the knock-out 
mutant BCG-65 (Lane 2) gave two bands at 4.8 kb and 1.7 kb resulting from 
the SacI site of the hyg gene integrated into the mce gene. The single 
cross-over mutant BCG-69 (Lane 3) gave three bands. 
Lanes 4 to 8 show the results of the XhoI digests. The wild-type strain 
(Lane 4) gave a single band at 4.7 kb while the knock-out mutants BCG-65 
(Lane 5), BCG-73 (Lane 7) and BCG-83 (Lane 8) gave a single band at 6 kb 
resulting from the presence of the hyg gene. The single cross-over mutant 
BCG-69 (Lane 6) gave two bands. 
Example 7 
This Example illustrates Western Blot analysis of the BCG transformants. 
M. bovis BCG transformants, prepared as described in Example 3, were grown 
in 10 ml of 7H9-ADC-Tw with 50 .mu.g/ml of hygromycin, to an optical 
density at 600 nm of 2. 1.5 ml of the culture was harvested, spun down 
(12000.times.g for 10 min) and transformants resuspended in 200 .mu.l of 
water. The solution was submitted to two 30 sec sonication cycles in a 
Sonifer 250 sonicator (Branson) at full power. The lysate was mixed with 
4.times.UMS buffer (0.1 M Tris-HCL, pH8; 20% glycerol; 8% SDS; 48% urea, 
8% .beta.-mercaptoethanol; trace of bromophenol blue). 8 .mu.l of the 
mixture was boiled for 10 min, resolved on a 12.5% acrylamide gel and 
transferred to a polyvinylidene fluoride membrane (Immobilon-P, 
Millipore). The membrane was processed using the Western Blotting system 
from Boehringer Mannheim, following the manufacturer's instructions. Mouse 
monoclonal antibodies against the mycobacterial cell entry protein (Mcep) 
were used for the blotting at a concentration of 1 .mu.g/ml. The 
anti-mouse horseraddish peroxydase-conjugated secondary antibody 
(Bochringer Mannheim) was used according to the supplier's 
recommendations. The blot was exposed to a film for 5 min at room 
temperature and the auto radiograph developed (see FIG. 4). 
FIG. 4 shows the Western Blot results. The monoclonal antibody to Mcep 
detected no production of Mcep by the knock-out mutant BCG-65 (Lane 1) 
while production of Mcep by both the single cross-over mutant BCG-69 (Lane 
2) and wild-type (Lane 3) was detected. 
Summary of the Disclosure 
In summary of this disclosure, the present invention provides mutants of 
Mycobacterium strains in which the expression of the mycobacterial cell 
entry protein is disabled. Modifications are possible within the scope of 
the invention. 
TABLE 1 
______________________________________ 
Sequence of PCR Primers 
PRIMER # SEQUENCE (5'-3') SEQ ID NO 
______________________________________ 
P4414 GTATGTGTCGTTGACCACGCC 
1 
P4448 TCAGGTCGATCGGCATCGTAGAAG 
2 
P4973 TTTCAAACGTTCCTGCGTCCC 
3 
P4974 CGAGTTTGACGATTCCAG 4 
______________________________________ 
REFERENCES 
1. Grange, J. M.; Gibson J; Osborn, T. W.; Collins, C. H. and Yates, M. D. 
(1983), Tubercle 64: 129-139. 
2. PCT; WO 95/17511, Jacobs, W. R. Jr.; Bloom B. R., Collins, D. M., 
Delisle, G. W.; Pascopella, L. and Kawakami R. P. 
3. Shepard, C. C. (1958), J. Exp. Med. 107: 237-45. 
4. Arruda, S., Bonfim, G.; Huma-Byron, T. and Riley L. W. (1993), Science 
261: 1454-1457. 
5. Azad, A. K., Sirakova T. D., Rogers L. M., Kolttukudy P. E. (1996) PNAS 
93: 4787-4792. 
6. Balasubramanicm V. M. et al (1996) J. Bacteriol 178:273-279. 
7. Reyrat J. M., Berthet F. X., Gicquel B. (1995) PNAS 92:8768-8772. 
8. Reyrat J M, Lopez-Ramirez G, Ofredo C, Gicquel B, Winter N. (1996), 
Urease activity does not contribute dramatically to persistence of 
Mycobacterium bovis bacillus Calmette-Guerin. Infect. Immun. 64. pp 
3934-3936. 
9. Jacobs Jr W R., Kalpana G V., Cirillo J D., Pascopella L, Snapper S B., 
Udani R A., Jones W., Barletta R G., Bloom B R. (1991) Genetic systems for 
Mycobacteria. Methods Enzymol. 204 pp 537-555. 
10. "Molecular Cloning: A Laboratory Manual", ed Sambrook. J.; Fritsch, E. 
F. and Maniatis, T. (1989) Cold Spring Harbour Laboratory Press. 
11. Anderberg, R. J., Strachan, J. A. and Cangelosis, G. A. (1995) Bio 
Techniques 18:217-219. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 4 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 21 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
#21 ACGC C 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 24 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
# 24CGTA GAAG 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 21 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
#21 GTCC C 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 18 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
# 18 AG 
__________________________________________________________________________