Note: Descriptions are shown in the official language in which they were submitted.
Wo 95117511 217 9 7 7 2 PCT/lIS94/14912
MYCOB~ KIA VIRULENCE F~CTORS ~ND A MEIHOD FOR THEIR IDENTIFICATION
Technical Field
This invention relate3 to polynucleotide
sequence(s~ associated with virulence in mycobacteria,
methods for isolating such sequence (s), and the use of
such sequence (s) in human and animal medical practice.
It also relates to polypeptides encoded in the sequence3.
Backqround Art
The mycobacteria are rod-shaped, acid-fa6t,
aerobic bacilli that do not form spores. Several species
of mycobacteria are pathogenic to humans and/or animals,
20 and determining factora associated with their virulence
are of prime importance. For example, tuberculosis is a
worldwide health problem which causes approximately
3 million deaths each year (17), yet little is known
about the molecular basis of tuberculosis pathogenesls.
25 The disease is caused by infection with Mycobacterium
tuoerculosis; tubercle bacilli are inhaled and then
ingested by alveolar macrophage9. As i5 the case with
most pathogens, infection with M. tuberculosis does not
always result in disease. The infection is often
3~ arrested by a developing cell---';At~ immunity (CMI)
resulting in the formation of microscopic lesions, or
tubercles, in the lung. If CMI does not limit the spread
of M. tuberculosis, caseous necrosis, bronchial wall
erosion, and pulmonary cavitation may occur. The factors
35 that determine whether infection with M. tuberculosis
results in disease are incompletely understood.
W095/17511 2,~9~ PCr/US94/14912 ~
The tuberculosis complex i8 a group of four
mycobacterial 3pecies that are 50 closely related
genetically that it has been proposed that they be
combined into a singl~ species. Three important members
5 of the complex are Mycobacterium tuberculosis, the major
cau3e of human tuberculosis; Mycobacteriu~21 africanurn, a
major cause of ~human tuberculo3is in some populations;
and Nycobacterium bovis, the cause of bovine
tuberculosis. None of these mycobacteria is restricted
lO to being pathogenic for a single host species. For
example, M. bovis causes tuberculosis in a wide range of
animals ;nr~ in r humans in which it causes a disease
that is clinically indistinguishable from that caused by
M. tuberculosis. Human tuberculosis i9 a major cause of
15 mortality throughout the world, particularly in less
developed countries. It accounts for approximately eight
million new cases of rl;n;r-l disease and three million
deaths each year. Bovine tuberculosis, a3 well as
causing a small percentage of these human cases, is a
20 major cause of animal suffering and large economic costs
in the animal industries.
Antibiotic treatment of tuberculosis i9 very
expensive and requires prolonged administration of a
combination of several antituberculosis drugs. Treatment
25 with single antibiotics is not advisable as tuberculosis
organisms can develop resistance to the therapeutic
levels of all antibiotics that are effective against
them. Strains of M. tuberculosis that are resistant to
one or more antituberculosis drugs are be n~ more
30 frequent and treatment of patients infected with such
strains is expensive and difficult. In a small but
increasing percentage of human tuberculosis cases the
tuberculosis organisms have become resistant to the two
most useful antibiotics, icrn;~7;r~ and rifampicin.
35 Treatment of these patients presents extreme difficulty
WO 95~1?511 217 9 7 7 2 PCr/US94/14912
and in practice is often unsuccessful. In the current
situation there is clearly an urgent need to develop new
- methods ~or detecting virulent strains of mycobacteria
and to develop tuberculosis therapies.
There is a recognized vaccine for tl~h~rr-lll osis
which is an att~n1~ted form of ~. bovis known as BCG.
This is very widely used but it provides incomplete
protection. The devP~ ' of BCG was completed in 1921
but the reason for its avirulence wag and hag rnnt;nll~fl
to remain unknown ~Grange et al ., 1983 ) . Methods of
~tt~n11~tin~r~ tuberculosis strains to produce a vaccine in
a more rational way have been investigated but have not
been successful for a variety of reasons ~Young, 1993).
Elowever, in view of the evidence that dead M. bovis BCG
was less ef~ective in conferring immunity than live BCG
~Block and Segal, 1955), there exists a need for
att,on~1~ted strains of mycobacteria that can be used in
the preparation of vaccines.
A variety of compounds have been proposed as
virulence f~actors for tuberculosis but, despite numerous
invest;~t;nnR, good evidence to support these proposals
is lacking. Nevertheless, the discovery of a virulence
factor or factors for tuberculosis is still regarded as
important and is a very active area of current research.
This is because such a discovery would not only enable
the possible development of a new generation of
tuberculosis vaccines but might also provide a target for
the design or discovery of new or improved anti-
tuberculosis drugs or therapies.
The ability to transfer and express
recombinant DNA among the myrnh~r~r~ia, first
demonstrated in 1987 ~Jacobs et al. ), enables the usage
of molecular genetics to elucidate pathogenic ~~~h~n;~ -
But, the present lack of evidence of homologous
3~ recombinatlon ill the pathogenic mycobacteria
WO95117511 2~ 9~ 2 PCr/US94/14912
has prevented the application of allele
exchange systems (Kalpana et al . ) for the
analysis .
One of the first examples of in vivo selection
5 for virulent bacteria was demonstrated by the classic
work of Grlffith et al. in 1928. Griffith using
~n~ CCi observed that, as a result of yenetic
exchange, virulent, capsulated pneumococci were recovered
from mice infected with a mixture of live attenuated,
10 non-capsulated pneumococci and heat-killed capsulated
rn~ cci ~Griffith, 1928) . However, similar systems
have not been demonstrated in mycobacteria.
Bacterial RNA polymerases are composed of a
core en~yme with the subunit composition ~2~' and one of
15 a variety of sigma factors. Transcription responses to
changes in growth conditions are modulated by multiple
RNA polymerases having different sigma factors which
promote transcription of different cIasses of promoters.
The principal sigma factor plays a central role in
20 bacterial by promoting -q,q~nti~l nhous-~kf~rin~" genes.
Genes for alternative sigma factors are present in all
bacteria and have been shown to promote specif ic
virulence genes in some pathogens ~Fang, 1992; Deretic
1994) . However, loss o~ a virulence phenotype due to
25 mutation in a principal sigma factor has not been
reported. Streptcrnyces sp. contain several homologues of
principal sigma factors ~Buttner, 1990) which are not
essential for normal growth but which appear to have a
function under certain growth conditions.
References cited in the Back~Trolln~ ~rt
1. Anon ~1972) TRT.lDEAU Myf~R~ Tr~rT~r~ CULTr~RE COLLECTION
(Trudeau Institute Inc., P.O. Box 59, Saranac ~ake, New
York 12 9 8 3 ) .
~7~
W09511751l 77~ PCT/US94/14912
2. Belisle, J.T., Pascopella, L., Inamine,
J.M., Brennan, P.J., and W.R. Jacobs, (l991) ~Isolation
- and expression of a gene cluster responsible for
biosynthesis of the glycopeptidolipid antigens of
Mycobacterium avium, " J. Bacteriol. 173 :6991-6997.
3. Bloom, B.R., Tuckman, M., Kalpana, G.V., and
W . R . Jacobs, l~nr--hl; ~h~ jl data .
4. Boyer, B., and D. Roulland-Dussoin, (1969)
"A complementation analysis of the restriction and
modification of DNA in Escherichia coli, ~ J. Mol. Biol.
41: 459 -472 .
5. Collins, D. M., and G. W. de Lisle, (1984)
"DNA restriction endonuclease analysis of lUycobacterium
tuberculosis and ~ycobacterium bovis BCG, " J. Gen.
Microbiol. 130:1019-1021.
6. Collins, F. M., and M. M. Smith, (1969)
"A comparative study of the virulence of Mycobacterium
tuberculosis measured in mice and guinea pigs, ~ American
Review of Respiratory Disease 100:631-639.
7. Collins, D. M., S. K. Erasmuson, D. M.
Stephens, G. F. Yates, and G. W. de Lisle, (1993) "DNA
fingerprinting of Mycobacterium bovis strains by
restriction fragment analysis and hybr~ 7~;nn with the
insertion elements IS1081 and IS6110, " J. Clin.
Microbiol. 31:1143-1147.
8. Dannenberg, A.M., Jr., (l991) "Delayed type
hypersensitivity and cell mediated immunity in the
pathogenesis of tuberculosis," Irununology Today 12:228-
233 .
9. Gallagher, J., and D. M. E~orwill, (1977) "A
selective oleic acid albumin medium for the cultivation
of Mycobacterium bovis, " J. Hyg. Camb. 79:155-160.
10. Grange, J. M., J. Gibson, T. W. Osborne, C.
E~. Collins and M. D. Yates, (19~33) "What is BCG?"
Tubercle 64:129-139.
WO95~17511 2~ ~1 9 ~ ~ 2 PCr/US94/~4912 --
11. Griffith F., (1928) "Significance of
pneumococcal types, n IJ, Hyg. 27 :113-159 .
12. Grn~k;~ky, C.M., Jacobs, W.R., Jr.,
Clark-Curtiss, .J.E., and B.R. Bloom, (1989) ~'Genetic
5 relationships among Mycobacterium leprae, Mycobacterium
tuberculo3i3, and candidate leprosy vaccine strains
determined by DNA hybridization: Identif ication of an M.
leprae-specific repetitive sequence, " Infect. Lmmun.
57: 1535, 1541.
13. Jacobs, W.R., Barrett, J.F., Clark-Curtiss,
J.E., and R. Curtiss III, (1986) "In vivo repackaging of
recombinant cosmid molecules for analysis of .SA7'_-7n~77A
typhimurium, Streptococcu3 mutans, and mycobacterial
genomic libraries, " Infect. Immun. 52 :101-109 .
14. Jacobs, W. R., G. V. Kalpana, J. D.
Cirillo, L. Pascopella, S. B. Snapper, R. A. Udani, W.
Jones, R. G. Barletta and B. R. Bloom, (1991) "Genetic
systems for mycr,h~rtP~i ~, " Method3 Enzymol . 204: 537-555 .
15. Jacobs, W.R., Tuckman, M., and B.R. Bloom,
20 (1987) "Introduction of foreign DNA into mycobacteria
using a shuttle plasmid," Nature 327:532-535.
16. Ralpana, G.V., Bloom, B.R., and W.R.
Jacobs, (1991) "Insertional m~lt~n~is and illegitimate
recombination in mycobacteria, " Proc. Natl . Acad. Sci .
USA 88: 5433 -5437 .
17. Cochi, A., (1991) "The global tuberculofiis
situation and the new control strategy of the World Heath
Organization, n Tubercle 72:1-12.
18. Dee, M.X., Pascopella, L., Jacobs, W.R.,
and C.F. Xatfull, (1991) "Site-specific integration of
mycobacteriophage L5: Integration-proficient vectors for
Mycobacterium. 3megmati3, Mycobacterium tuberculosis, and
bacille Clamette-Guerin, " Proc. ~atl . Acad. Sc~ . U.S.A.
88: 3111-3115 .
~ WO95/17511 ~ 79772 PCr/US94/14gl2
19. r~ , G.B., Smith, N., and A.Q. Wells,
(1954) "The growth of intracellular tubercle bacilli in ~=~
relation to their virulence, " Am. Rev. Tuberc. 69 :479-
494 .
20. North, R.J., and A.A. Izzo, (1993)
"Mycobacterial virulence: Virulent 3trains of
~yco~acterium tuberculosis have faster in vivo doubling
times and are better equipped to resist growth inhibiting
functions of the maL~ ha~s in the presence and absence
of specific immunity, " J. Exp. Med. 177:1723-1734.
21. Oatway, W.H., Jr., and W. Steenken, Jr.,
(1936) "The pathogenesis and fate of tubercle produced by
dissociated variants of tubercle bacilli, " ~J. In~. Dls.
59 :306-325 .
22. P~COP~ L., F. M. Collins, J. M.
Martin, M. H. Lee, G. F. Hatfull, B. R. Bloom and W. R.
Jacobs, "In vivo complementation in Mycobacterium
tuberculosis to identify a genomic fragment associated
with virulence, " Infection and Immunity 62: 1313-1319.
23. Pierce, C.H., Dubos, R.J., and W.B.
Schaefer, (1953) "Multiplication and survival of tubercle
bacilli in the organs of mice," J. Exp. Med. 97:189-206.
24. Sambrook, J., E. F. Fritsch and T.
Maniatis, (1989) MOLECULAR CLONING: A LABORATORY MANUAL (Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY).
25. Steenken, W., Jr., and L.U. Gardner,
(1946 . ) "History of H37 strain tubercle bacillus, " Amer.
Rev. ~uberc. 54:62-66.
26. Steenken, W., Jr., Oatway, W.H., Jr., and
S.A. Petroff, (1934) "Biological studies of the tubercle
bacillus. III. Dissociation and pathogenicity of the R
and S variants of the human tubercle bacillus (H37), " .J.
Exp. Med. 60:515-540.
27. Stover, C.K., de la Cruze, V.F., Fuerst,
T.R., Burlein, J.E., Benson L.A., Bennett L.T., Bansal,
WO 95/17511 . ,~ .., r PCT/US94/14912
2~79~2
G.P., Young, ~.F., ~ee, M.H., Hatful, G.F., Snapper,
S.3., Barletta, R.G., Jacobs, W.R., and B.R. Bloom,
(1991) "New use of BCG for recombinant vaccines, " Nature
351: 456-460 .
28. van Soolingen, D., P. W. M. Hermans, P. E.
W. de Haas, D. R. Soll and J. D. A. van Embden, ~1991)
"Occurrence and stability of insertion sequences in
Mycobacterium.. tuberculosis complex strains: evaluation
o~ an insertion sequence-dependent DNA polymorphism as a
tool in the epi~i~m;Qlo~y of tuberculosis, " J. Clin.
Microbiol. ag:2578-2586.
29. Weis, H., ~1991) CURRENT EIROTOCOLS IN MOLECUI~A~
~IOLOGY~ supplement 13, 5.3. (Greene pl'h~ nS
Associates , New York , eds ., F . M . Ausubel , R . Brent , R .
E. Kingston, D. D. Moore, J. G. Siedman, J. A. Smith, and
K . Struhl ) .
30. Young, D. B. and S. T. Cole, (1993)
"~eprosy, tuberculosis, and the new genetics, "
~J. Bacteriol. 175:1-6.
31. Block, H. and W. Segal, Am. Rev. Tuberc.
Pulm. Dis 71:228-248.
32. Fang, C.F. et al., (1992) Proc. Natl.
Acad. Sci. USA 89:11978-11982.
33. Deretic., V. et al, (1994) J. Bact.
176:2773-2780.
34. Buttner, M.J., et al., (1990) J. Bact.
172: 3367-3378 .
SummarY of the Invention
The present invention provides i ~Al AtF~l and
re~ '-; n~nt polynucleotide sequences associated with
virulence detlormin~nt~ in members of the genus
my~-rh~- t~ria, particularly those of ~the tuberculosis
complex, and more particularly in ~. tuberculosis and M.
35 bovis. Based upon homology to sigma factors from other
W095117511 772 PCr/US94114912
microorganisms, one of the mycobacterial sequences
associated with virulence encodes a putative sigma-like
f actor .
The DNA sequences ~nrrrl;nr; factors associated
with virulence were found by the use of in vivo
tion assays, more particularly by
complc tat i nn n a guinea pig model and in a mouse
model . The in vivo genetic compl:~ t~t; on systems
utilized integrating shuttle cosmid libraries to identify
potential virulence ge~es. Thus, the invention also
provides techniques to identi~y a DNA serluence or
sequences associated with virulence determinants in M.
tuberculosis and M. bovis and similar DNA sequences in
other tuberculosis complex strains and in strains of
other mycobacterial species and in species of other
pathogenic organisms.
Accordingly, Pmhofi; tR of the invention
include the following.
A method for identifying a DNA sequence or
sequences associated with virulence det~r-m;n~nt~ in M.
tuberculosis and M. ~ovis and similar DNA sequences in
other tuberculosis complex strains and in strains of
other mycobacterial species and in species of other
pathogenic orrJ:ln;, comprising the steps of:
a) preparing a genomic DNA library of the
pathogenic organism;
b) constructing an int~s~atinrJ shuttle vector
rnntioin;nrJ genomic inserts ~Le:~dIed in step a);
c) transforming via homologous rec' ' in~tion a
3 0 population of avirulent organisms;
d) isolating the recombinants;
e) inoculating a subject with an adequate
inoculum of the recombinants in order to select virulent
recombinants;
f ) isolating the virulent recombinants; and
Wo 95/17~ 9~ PCrlUS94114912
g) identifying the DNA insert which confers
virulence .
Thi6 method may be performed with individuals
that are mice or guinea pigs.
An isolated polynucleotide comprised o~ a
segment of le3s than 3kb that i5 f~ n~ y homologous
to a mycobacterial DNA se~auence ;~sQr~ ~te~l with virulence
in mycobacteria, whereln the myrnh::~tF~rial DNA sequence
encodes a sigma factor.
lo An isolated polynucleotide comprised of a
segment of less than 3 kh that encodes a polypeptide or
fragment thereof, wherein the polypeptide is associated
with virulence~in mycobacteria and is a sigma factor.
The polypeptide may be f~F~s~nti~11y homologous to the
polypeptide encoded in Figure 9.
An isolated polynucleotide comprlsed of at
least 15 sequential nucleotides homologous to a ser~uence
of polynucleotides in Figure 9.
A recombinant polynucleotide comprised of a
sequence of at least 15 sequential nucleotides homologous
to a sequence of polynucleotides in Figure 9.
A recombinant polynucleotide comprised of a
segment of less than 3 kb that encodes a polypeptide or
fragment thereof, wherein the polypeptide is associated
with virulence in mycobacteria and is a sigma factor.
An expression vector comprised of the
recombinant polynucleotide described above.
An isolated polynucleotide comprised of a
linear segment of at least 15 nucleotides that is
subs~nt;~lly homologous to myc~h~rt~rial DNA in
a plasmid s~ler~ from the group consisting of p~JHA1,
pUHA2, pUHA3, pl~HA4, pTlHA5, pUHA6, p~HA7, pl~HA8, pU~Ag,
pUHA11, pYt~}3352, pY~3353, and pYU~3354.
W09S/17511 217~772~ PCrAJS94/14912
A host cell comprised of any of the above-
described isolated polynucleotides, including expression
vectors .
A diagnostic kit comprised of a polynucleotide
and a buffer packaged in suitable vials, wherein the
polynucleotide is any of the above-described isolated
polynucleotides .
An isolated polypeptide gubst~nt;~lly
homologous to a polypeptide associated with virulence in
mycobacteria or a f ragntent thereof, wherein the
mycobacterial polypeptide is a sigma factor. The
mycohArt~rial polypeptide may be one that is encoded in a
DNA sequence shown in Figure 9.
An isolated polynucleotide comprised of a
segment of less than 3kb that is essentially homologous
to a mycobacterial DNA sequence associated with
avirulence irt mycobacteria, wherein the mycrh~rt~rial DNA
sequence encodes a sigma factor.
A method for producing an altered property in a
wild-type bacterial strain other than M. bovi~ comprising
mutagenizing a principal sigma factor in the bacteria,
wherein the mutagenizing results in converting an
arginine to a histidine in the principal 8igma factor,
and wherein the conversion occurs at a similar position
to that present in ~. bovis ATCC 35721. This method
includes altering the virulence properties of the
bacterial strain.
A method of using a bacterial strain prepared
by the method described above, the method comprising
3 0 preparing a vaccine by mixing a pharmacologically
effective dose of the strain with a phar--rett~1r~11y
- acceptable suitable excipient.
11
Wo 95117511 ~ 2 PCT~S94/14~12
Brief Descri~tion of ~h-~ Drawin~s
Figure 1 i8 a schematic illustrating the
strategy for recovering part of cosmid plJHAl from IY.
bovis WAg300 which i8 a member of the ~. bovis
ATCC35721 (pYU}3178: :M. bovis WAg200) library and which has
increased virulence for guinea pigs. The diagrams are
not to scale.
Figure 2 is a schematic showing the alignment
of p~HA2-pUHA7 in linear form for comparison purposes
b~r,;nninJr with the NotI site at position 2024 of pYUB178.
Cosmids p~A3-PUHA7 were isolated by colony hybridization -
using a probe of the 2 kb MluI fragment of P~}~A2: M, MluI
site; N, NotI site;
c, vector arm; , insert DNA from
M. bovis Wag200.
Figure 3 is a restriction map of cosmid PUEIA3
in linear form starting with the NQtI site at position
2024 of pYUB178:h, NheI; M, MluI; N, NotI, X, XbaI.
Figures 4A-C represent a map of the integrating
shuttle cosmid, pYUB178, and analysis of individual
clones and pools of H37Ra (pYUBl78: :H37Rv) .
Figure 4A shows the ~ ~ ~ntc that allow
integration of pYUB178 into the mycobacterial genomes are
attP and int. The pY~3178 cosmid rrnt:~;nR an E~. coli
ori, the L5 attP, the I-5 int, a kanamycin resistance
gene, aph, derived from Tn903, lambda cos, and a uni~r,ue
cloning s i te, Bcl I .
Figure 4B ia a schematic showing identif i r~ ir~n
of the pYUB178/H37Rv junctional fragments within the
chromosome of a H37Ra recombinant rrnt:,ininrJ
pYUB178: :H37Rv DNA. PstI-digested .,h~ E 1 DNA is
separated by gel electrophoresis and hybridized with a
labeled probe from pYUB178. The probe is the 1.1 kb
DraI/SspI DNA fragment of pYUB178 that flanks the 3clI
cloning site. The integrated pYUB178: :H37Rv cosmid can
12
W095/17511 21~7~ PCr/US94/14912
be detected only by the presence of pYHB178-hybridizing
DNA fragments. The PstI sites on either side of the
H37Rv insert are fixed. Thus, the size of hybridizing
DNA fragments varies with the H37Rv insert DNA.
Figure 4C are half-tones of gels showing
individual H37Ra recombinants r~nt~;n;n~ pYUB178: :H37Rv
cosmid clones were i ~ol at~Cl from mouse lung tissue after
spleen passage of recombinant pools, experiment J5P (see
Table 9) . Pools of H37Ra(pY~B178: :H37Rv) were collected
and passaged in broth culture. The chromosomal DNAs from
pools and individual clones were isolated, digested with
PstI, separated by agarose gel elec~rophoresis and
transferred to a nylon filter to hybridize with the 1.1
kb DraI/SspI DNA fL _ of pYUB178. ~anes 1-3, the ---
H37Rv DNA j1lnrt;~m~1 fragments of in vivo-selected
individual clones of pool 2; lanes 4 and 5, the H37Rv DNA
junctional f L _ ~ 8 of members of pool 3, before (lane
4) and after (lane 5) in vitro passage.
Figures 5A-B shows the growth of in vivo-
selected H37Ra (pYU3178: :H37Rv) clones in mouse lung and
spleen. Growth rates of clones mc2806, X37Rv, and mc2816
were measured and compared. The growth rate of mc2806 is
represented by solid squares on the solid lines, the
growth rate of mc2816 is represented by the open circles
on the dotted lines, and the growth rate of H37Rv is
represented by solid triangles on the dotted lines.
These data are representative of three experiments. See
text and Table 9, experiment ~J33, for experimental
details .
3 0 Figure 5A shows growth in spleen .
Figure 5B shows growth in lung.
Figures 6A-B illustrate the retrieval of H3 7Rv-
C~7nt~;n;n~ cosmids from the mc2806 c~
- Figure 6A i3 a schematic illustrating the
strategy used to retrieve the H37Rv insert DNA from the
-
W095/17511 2i~97~` PCrnJS94114912 ~
integrated cosmids in H37Ra (pYUB178: :H37Rv) recombinant~ .
Figure 6B i8 a half-tone of an autoradiograph
showing a Southern hybridization of A~eI and EcoRI
digests of mc2806 cl~ ~ 1 DNA, or cosmid DNAs that
were retrieved from the chromosome of mc28Q6. The 436 bp
AseI/~3clI fragment of pYl~B178 that Cnrlt:~;n~ cos was used
as a probe. ~ane 1, mc2806 c~ _ 1 DNA, lanes 2 to
17, DNA from sixteen individual retrieved cosmids.
Figure 7 is a graph showin~ the growth of H37Ra
recombinants cnnt;~n;n9' pYUB352-overlapping and -
nonoverlapping cosmids. H37Ra was separately transformed
with pYUB352-overlapping cosmids, pY17B353 and pYUB354,
and with unrelated cosmids, pYUB355 and pYU~3356. Growth
of each re~omh; n;lnt was measured over a time course in
mouse spleen. See Table 9, experiment J36. The growth
of pYU8353- a~d pYUB354-rnnt~in;ng H37Ra recombinants is
represented by the small squares on the solid lines. The
growth of mc2806 is represented by the large sguares on
the solid lines. The growth of pYtJB355- and p~B356-
cnnt~;n;n~ H37Ra recombinants is represented by the small
circles on the solid lines. The growth of mc2816 is
represented by the large circles on the dotted lines.
The growth of H37Rv is represented by the triangles on
the dotted lines.
Figures 8A-C represent the restriction map of
the ivg region of H37Rv DNA in pYUB352-overlapping
cos~ids. Restriction digests of pY~3352, pYUB353, and
pY~8354 were performed with EcoRI and ~indIII.
Figure 8A i8 a half-tone reproduction of gels
showing digested DNA ~- _ t8 which were separated by
agarose gel electrophoresis.
Figure 8B is a half-tone reproduction of gels
showing DNA fragments which were hybridized to the AseI
fragment of pYU;3352 that included its entire H37Rv insert
with flanking pYUB178 DNA sequences. The arrows point to
14
~ WO95/17511 1 79 7 PCrlUS94/14912
7~
DNA fragments that hybridize to pYU~178 DNA probes.
These bands are junctional fragments. ~anes 1-3 are ~~:
digests of pYUB352, lanes 4-6 are digests of pYUB353, and
lanes 7-9 are digests of pYUB354. I,anes 1, 4, and 7 show
EcoRI digestion patterns, lanes 2, 5, and 8 show EcoRI
and HindIII double digestion patterns, and lanes 3, 6,
and 9 show HindIII digestion patter~s.
Figure 8C is a 5~1- tic illustrating data
gathered from these molecular analyses and the functional
analyses shown in Figure 7 allowed the construction of
the physical map of the ivg region of EI37Rv that is
present in cosmids p~UB352, pYUB353, and pYUB354.
A=AseI, E=l~coRI, ~I=HilldIII.
Figure 9 and 9a is comprised of four sheets.
Figure 9 shows the nucleotide sequence of the coding
strand of the 2745 bp ~L _ t that restores virulence to
M. bovis ATCC35721. Figure 9a shows the same as in
Figure 9 together with a 530 amino acid sequence
translated from the largest ORF.
Figure 10A is comprised of two sheets showing
the results of a PileUp comparison of known principal
sigma factors from St ~ e~- yces coelicolor (GenBank
Accession Nos. %52980, X52981, x52983) and Streptomyces
griseus (GenBank ~c~sir~n No. LO8071) with the
translation of the largest ORF of the 2000 bp contig from
the M. bovis virulence restoring factor, rpoV, that
restores virulence to ~. bovis ATCC35721.
Figure 11 presents the results of a GAP
comparison of Streptomyces griseus principal sigma factor
(Peptide translation of GenBank accession No. LO8071 from
nucleotide numbers 570 to 1907, which is the coding
sequence of the hrdl3 gene) with peptide translation of
the large ORF of the appr-~Yi~-t-oly 3 kb DNA fragment from
- M. bovis associated with virulence.
WO 95/17511 2 1~ 9~ ~ 2 PCr/US94/14912 ~
Figure 12a-1 and 12a-2 (SEQ ID NO:13 and SEQ ID
NO :14 ) is comprised o~ two sheets showing the large ORF
of the M. boViB WAg200 se~uence which begins with GTG at
position 835-837.
Figure 12 (SEQ ID NO:8 through SEQ ID NO:12)
presents a comparison of putative principal sigma ~actors
of three kr. tub~rculosi~ complex strains and two
StreptomyceEI 8p.
Detailed Descril~tion of the Tnvention
The practice of the pre8ent invention will
employ, unless otherwise indicated, conv~n~ nAl
technilr,ues of molecular biology, microbiology,
re ' inAn~ DNA, and immunology, which are within the
skill of the art. Such techniriues are ~ l A;n~ fully in
the literature. See e.g., Sambrook, Fritsch, and
Maniatis, ~rnT.~TTT.~R CLONING: A L~BORATORY M~NUAL, Second
Edition (1989), OLIGONUCLEOTIDE ~ NL~ ; (M.J. Gait Ed.,
1984), the series METHODS IN ENZYMOI,OGY (Academic Press,
Inc . ); GEN-E TRANSFER VECTORS FOR MP.MM~T.T~r~T CELLS (J.M.
Miller and M.P. Calos eds. 1987), HANDBOOK OF
EXPERIMENTAL I~NOLOGY, (D.M. Weir and C.C. Blackwell,
Eds. ), CURRENT PROTOCOLS IN r/r~r~cTTT ~T~ BIO~OGY (F.M.
Ausubel, R. Brent, R.E. Ringston, D.D. Moore, J.G.
Siedman, ,T A. Smith, and R. Struhl, eds., 1987), and
CURRENT PROTOCOLS IN IMM[~NOLOGY (J.E. Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober,
eds., 1991l. All patents, patent applications, and
publications irn~-l herein, both f~upra and infra, are
3 0 incorporated herein by ref erence .
The present invention provides polynucleotides
that are associated with virulence in members oî the
gerLus myr~har~ria, and particularly in members of the
mycobacterial complex. Virulence is the relative
capacity of a pathogen to UV~L~ - body defenses; it is
16/l
RECTIFIED SHEET (RULE 91 )
ISA/EP
o9SJ17511 2179772 PCr/US94/14912
al o the relative ability to cause disease in an infected
host. In gram-negative bacterial pathogens, virulence i8
16/2
RECTIFIED SHEET (RULE 91 )
ISA/EP
Wo 95117511 Pcrl~7S94/14912
2~9~2
generally determined by a multiplicity of traits that
endow the pathogen with its ability to exploit anatomical
weaknesses and overcome the immune def enses o~ the host .
It is expected that a similar multiplicity of traits
5 determines the virulence of pathogenic mycobacteria.
Properties associated with virulence in microorganism~
include those listed in Table l.
T~hle l. Prs~erties ~R~ociated Witll virulence
l. Infectious; capable of being spread from
one individual ~o another.
2. Capable of entering ~ n host cells.
3. Capable of surviving or escaplng phagocyte
cellular defenses.
4 Capable of multiplying in host cells.
5 Capable o~ spreading ~rom one inEected
cell to an uninf ected cell .
6 Capable of causing cell in~ ury that
results in pathology.
In addition, a virulent organism may be capable o~
killing the inf~ected host.
sy mycobacteria is meant t~e genus that
includes the species M. phlei, M. smegmatis, M.
25 africanum, M. fortuitum, M. marinum, M. ulcerans, M.
tuberculosis, M. bovis, M. microti, M. avium, M.
paratuberculosis, M. leprae, M. lepraemurium, M.
intracellulare, M. scrofulaceum, M. xenopi, M. genavense,
M. kansasii, M. simiae, M. szulgai, M. ha; ~1 ~i7um, M.
30 asiaticum, M. malmoense, and M. shimoidei 0~ particular
interest are the members o~ the tuberculosis complex,
including M. tuberculosis, M. bovis, M. africanum and M.
mi cro ti .
As used herein, the term "virulence ~actor
35 ~nrrf~in~ seriuence~' denotes a polynucleotide sequence that
encodes a product that is associated with virulence in a
~ Wo95/17~11 21 7977,~ PCr/llSs4/14912
member of the mycobacterial species. This term is
r-nr ~-ARed within the term a "sequence associated with
virulence~ that denotes that a polynucleotide sequence
that confers a trait associated with virulence on an
5 avirulent mycobacterium, whether or not the
polynucleotide encodes a product. In particular, the
virulence a3sociated ~r~ nrl~ of the present invention
are those that confer one or more traits associated with
virulence and have a high degree of homology, i . e ., at
10 least about 7096 overall homology, preferably at least
about 80~ overall homology, even more preferably at least
about 90?~ overall homology, to the mycobacterial
polynucleotides described herein. Methods of determining
homology between sequences are known in the art, and
15 include, for example, direct comparison of sequences, and
hybri f~ i 7Zl t ir~n assays .
The sequence of one of the mycobacterial DNAs
nc;Atod with virulence, isolated from ~. bovi~, is
shown in Figure 9. This DNA rr,nt~;n~ several contigs and
20 an open reading frame (ORF) that based upon amino acid
sequence homology in certain regions, encodes a
polypeptide that i5 a putative sigma factor. Portions
or all of fragment of which the ORF is part is in
plasmids p~lHAl, pUE~A2, pUHA3, paHA4, p~A5, pUH~6, PUHA7,
25 pUHA8, p~HA9, or pUE~All. A particular Pmho~;rlrnt of the
invention is an isolated or recombinant polynucleotide .
that is comprised of all or segment of the ORF r-nrnfl;nrJ
the sir,ma factor.
Virulence is also associated with the
myrnhArt~r;Al sequences present in pYU}3352, pYln33~3, and
pYU3354. Thus, the isolated and r~c ' in:lnt
polynucleotides may also be comprised of sequences
homologous to the mycobacterial DNA in these plasmids.
The DNA sequences upon which the
3 5 polynucleotides of the invention are based were obtained
18
W095/17511 ~g'l~ Pcrluss4rl49l2
by the use of in vivo virulence complementation as3ays.
A method for identifying virulence df~t~orm;n~"tR by
genetic complementation in vivo was discovered that
requires: (i) t~qo strains that are genetically similar;
5 (ii) a phenotype associated with virulence; and
(iii) gene transfer systems,
Cosmid genomic libraries of virulent
mycobacterial strains of M. tuberculosis and M. bovis
were constructed in an integrating cosmid vector. An
10 example of an integrating cosmid vector is pYU~17~,
described by Lee et al. (1991), Proc. Natl. Acad. Sci.
U5A, ~ :3111-3115 and Pascopella et al. (1994), Infect.
Immun. 62:1313-1319-. The integrating vector,
approximately 5 kb long, can accommodate 40-45 kb of DNA
15 and uses the site-speciic integration system of
mycobacteriophage ~5 to integrate recombinant DNA into a
unique attB site of the mycobacterial hll ~ . This
vector thus can represent more than 95~ of the entire
mycobacterial genome in as few as about 300 clones. The
20 recombinant DNA introduced in single copy is stably
---int:~inf.r7 in mycobacterial cells in the absence of
antibiotic selectio~, even when the strain is passed
through animals. Thus, use of this vector reduced the
number of clones that needed to be screened, and ensured
25 that cloned genes were not lost during animal passage.
The genomic libraries in the integrating cosmid
vector were introduced into corresponding avirulent
strains of mycobacteria. Methods of introducing
polynucleotides into cells are known in the art, and
30 include, for example, electroporation, tr~nR~l~mt~rn and
transformation. In order to select for virulent
myrr-h:l~ t,~ria the resulting libraries of recombinant
clones were injected into animals, i.e., mice or guinea
pigs. It is thought that clones that restore virulence
35 may have a selective advantage and thus be enriched for
19
`~ WO95/17511 ~72~ PCr/US94114912
in the injected animals. In the mouse ~ m~ntation
assay, avirulent mutants cause a self-limiting infection
while virulent myroh~ct.orial strains multiply more
rapidly, and in high challenge doses cause death.
5 Similarly, in the guinea pig compl~ -~t;~n asgay,
avirulent mutants cause a self-limiting infection.
However, virulence in guinea pigs can be assessed by the
sites in which gross lesions are found. When avirulent
strains of mycobacteria are inoculated subcutaneously in
l0 a flank, these strains are not sufficiently virulent to
pass through the lymph nodes draining the inj ection site
and enter the systemic cirr~ t; nn in suf f icient numbers
to cause gross lesions to occur in the spleen. This is
contrasted to virulent strains, which under the same
15 inoculation conditions do give rise to spleen (and lung)
lesions. Examples of assay systems for comparing
avirulent and corresponding virulent mutants of N.
tuberclllosis and M. bovis are described in the Examples.
Clones of mycobacteria that had been rendered
20 virulent by the integration of a polynucleotide encoding
a virulence f actor were isolated . Portions of the ---
integrated virulence determining cosmid were isolated
from the clones by restriction enzyme digestion, and the
f ragments were reinserted into the integrating vector and
25 assayed for virulence factor activity using in vivo
compl -~tirr assays. These assays led to the
nt; f; cation of mycobacterial DNA encoding polypeptides
associated with virulence. In the case of ~. ~ovis, the
sequence of a f ragment of mycobacterial DNA of
30 apprrl~ir-t--ly 3 kb in a clone designated p~HAll was
determined. A comparison of Gen;3ank sequences with the
amino acids encoded in the fr~; t, and particularly
within a large ORF and an ad~acent contig, showed a
significant degree of homology with sigma factors from
35 other mi~L~,~,L~ isms, indicating that the large ORF
WO 95117511 ~ PCr/US94114912
encodes a putative sigma factor. On the basifi of this
homology and the ability of the WAg200 gene to confer a
virulence phenotype we have named the gene, rpoV. The
high degree o homology between the principal sigma
5 factors of St~eptomyces sp. and the putative 3igma
factors from the M. tu~erculosis complex may reflect
their evolutionary rpl~ti~n~hip and the fact that both
theae genera have DNA with a high guanine plus cytosine
percentage .
A comparison of the homologous DNA sequences
from M. bovis WAg200 and the DNA sequence from the
att.-nl~t--rl M. bovis ATCC35721 indicated that the latter
had no sequence differences upstream of the ORF but had
two point differences in the coding sequence. One of
these diferences was also present in the virulent strain
M. tu}~erculosls Erdman but the other di~erence, which
caused an arginine to histidine change at position 522,
was not found in any of the virulent strains analy2ed.
Thus we deduce that this is the likely mutation that
causes M. ~ovis ATCC35721 to become avirulent. This
position is highly conserved among principal sigma
factors and their homologues and the region in which it
occurs ha3 the characteristics of a helix-turn-helix
motif and is believed to be involved in -35 sequence
recognition. See l.onetto, M., Gribskov, M. and Gross,
C.A., (1992) J. Bact. 174: 3843-3849. Thus, as used
herein, the term '~similar position to that present in M.
~ovis ATCC35721" in reference to arginine to histidine
conversion in a bacterial strain with a mutagenized
principal sigma factor contemplates one in a region that
is highly conserved among principal sigma factors and
their homologues and one that has the characteristics of
a helix-turn-helix motif and i5 believed to be involved
in -35 sequence recognition.
21
Jl~ WO95/17511 217~7772 PCT/US94114912
While the virulence assays initially were used
to isolate the polynucleotides described herein, they may
also be used to determine whether polynucleotides
constructed from the information and sequences provided -
5 herein and factors transcribed and/or translated
therefrom are associated with virulence in mycobacteria,
and particularly in ~. bovis or M. tuberculosIs.
One F~mhr~; t of the invention is an isolated
polynucleotide comprised of a sequence associated with
l0 virulence in mycobacteria. Another embodiment of the
invention is an isolated polynucleotide comprised of a
8equence associated with avirulence in mycobacteria. As
used herein the term "polynucleotide~ refers to a
polymeric form of nucleotides of any length, either
15 ribonucleotides or deoxyribonucleotides. This term
ref ers only to the primary structure of the molecule .
Thus, this term includes double- and single-stranded DNA
and RNA . It also includes known types of modif ications,
for example, labels which are known in the art (e.g.,
20 Sambrook, et al . ), methylation, "caps", substitution of
one or more of the naturally occurring nucleotides with
an analog, ;nt~rn~ eotide modifications such as, for
example , those with uncharged linkages ~ e . g., methyl
rh~s~hnn~tes~ phosphotriesters, rh~sphr~m;dates,
25 carbamates, etc.~, those cont~;n;nS pendant moieties,
such as, for example, proteins (including ~or e.g.,
nucleases, toxins, ~ntiho~;es~ signal peptides, poly-~-
lysine, etc. ), those with intercalators (e.g. , acridine,
psoralen, etc.), those ~rmt~;n;ng h~l~t-~rs (e.g.,
30 metals, radioactive metals, boron, oxidative metals,
etc.), those c-~nt~;n;n~ alkylators, those with modified
linkages ( e . g. , alpha anomeric nucleic acids , etc. ), as
well as unmodified forms of the polynucleotide.
Polynucleotides include both sense and antisense
35 strands. Recombinant nucleic acids comprising sequences
WO95117511 ~ PCrlUss4/149l2
otherwise not naturally occurring with the designated
mycobacterial sequence are also provided by this
invention. Although the wild type sequence may be
employed, the wild type sequence will often be altered,
5 e . g ., by deletion, substitution, or insertion .
The nucleic acid sequences used in this
invention will usually compri6e at least about 5 codons
~ 15 nucleotides l, more usually at least about 7 to 15
codons, and most preferably at least about 35 codon~.
10 One or more introns may also be present. Thia number of
nucleotides is usually about the minimal length required
for a successful probe that would hybridize specifically
with such a sequence.
Techniques for nucleic acid manipulation are
15 described ~PnP~Rlly, for example, in Sambrook et al.,
~,, or Ausubel et al., ~. Reagents useful in
applying such technique3, such as restriction enzymes and
the like, are widely known in the art and commercially
available rom such vendors as New England sio~abs,
20 Boehringer M~nnhPirn, Amersham, Promega Biotec, ~J. S.
Bio~hPmi~-Rl~, New England Nuclear, and a number of other
sources .
The polynucleotides of the invention will have
substantial homology or similarity to the DNAs di3closed
25 herein that are associated with virulence or with
avirulence in mycobacteria. A nucleic acid or fragment
thereof is ''substAn~iAlly homologous~ ~or "substantially
similar" ) to another if, when optimally aligned (with
appropriate nucleotide insertions or deletions) with the
30 other nucleic acid (or its complementary strand), there
is nucleotide sequence identity in at least about 60~ of
the nucleotide bases, usually at least about 70~, more
usually at least about 809~, preferably at lea3t about
90~, and more preferably at least about 95 to 989~ of the
35 nucleotide bases.
23
WO95/17511 2I7~1772 PCrlUS94/14912
Alternatively, a nucleic acid or fragment (or
its complementary strand) is substantially homologous (or
similar) with a DNA associated with virulence or with
avirulence in mycobacteria when they are capable of
hybridizing under selective hybridization conditions.
Selectivity of hybridization exists when hybridization
occurs which is subst~nt;~lly more selective than total
lack of specificity. Typically, selective hybr;~i7sit;~n
will occur when there is at least about 65~ homology over
a stretch of at least about 14 nucleotides, preferably at
least about 70%, more preferably at least about 75%, and
most preferably at least about 909~ . See, 7~z~nPhi ~A (1984)
Nuc. Acids Res. 12 :203-213 . The length of homology
comparison, as described, may be over longer stretches,
and in certain embodiments will often be over a stretch
of at least about 17 nucleotides, usually at least about
20 nucleotides, more usually at least about 24
nucleotides, typically at least about 28 nucleotides,
more typically at least about 32 nucleotides, and
preferably at least about 36 or more nucleotides.
Nucleic acid hybridization will be af f ected by
such conditions as salt concentration (e.g., NaCl),
temperature, or organic solvents, in addition to the base
composition, length of the complementary strands, and the
number of nucleotide base mismatches between the
hybridizing nucleic acids, as will be readily appreciate~
by those skilled in the art. Stringent temperature
conditions will generally include temperatures in excess
of 30 C, typically in excess of 37, and preferably in
excess of 45. Stringent salt conditions will ordinarily
be less than 1000 mM, typically less than 500 mM, and
preferably less than 200 mM. ~Iowever, the combination of
parameters is much more important than the measure of any
single parameter. See, e.g., Wetmur and Davidson (1968)
J. Mol. ~3iol, ~L:349-370.
24
-
Wo95117511 ~9~ ~2 PCr/US94114912
The polynucleotides of the invention are
isolated or sub3tantially purified. An ~isolated~' or
"subst~nt;~lly pure" or "purified" nucleic acid i6 a
nucleic acid, e.g., an RNA, DNA, or a mixed polymer,
5 which is subst~nti~lly separated from other mycobacterial
^-,t c that naturally accompany the sequences
associated with virulence, e.g., ribosomes, polymerases,
and many other mycobacterial polynucleotides such as RNA
and other O~ 1 sequences. The term embraces a
lO nucleic acid seriuence which has been removed from its
naturally occurring environment, and includes recombinant
or cloned DNA isolates and chemically synthesized
analogues or analogues biologically synthesized by
heterologous systems.
The term "recombinant polynucleotide" as used
herein intends a polynucleotide of genomic, cD~A,
semisynthetic, or synthetic origin which, by virtue of
its origin or manipulation: (l) is not associated with
all or a portion of a polynucleotide with which it is
20 associated in nature; or (2) is linked to a
polynucleotide other than that to which it i9 linked in
nature; and (3) does not occur in nature. This
artificial combination is often ~r, ~ h~rl by either
chemical synthesis means, or by the artif;rj~l
25 manipulation of isolated segments of nucleic acids, e.g.,
by genetic rn~i n~ri n~ techniques . Such is usually done
to replace a codon with a r~ n~l~nt codon encoding the
same or a conservative amino acid, while typically
introducing or removing a ser~uence recoJnition site.
30 Alternatively, it is performed to join together nucleic
acid segments of desired functions to generate a desired
rnmhin~t'nn of functions.
In some ~ of the invention the
polynucleotides encode a polypeptide associated with
35 virulence or with avirulence A nucleic acid is said to
Wo 95117511 21 79 7 72 PCr/US94/14912
~'encode~ a polypeptide ii-, in its native state or when
manipulated by methods well known to those skilled in the
- art, it can be transcribed and/or translated to produce
~he polypeptide or a fragment thereof. The anti-sense
- 5 strand o~ such a nucleic acid i8 also said to encode the
sequence .
Also rrnt ,1~Ated within the invention are
expression vectors comprised of a sequence encoding a
polypeptide associated with virulence. Expression
vectors generally are replicable polynucleotide
constructs that encode a polypeptide operably linked to
suitable transcriptional and translational rerulatory
elements. Examples of regulatory elements usually
included in expression vectors are promoters, ~nhAnrPrs,
ribosomal binding sites, and transcription and
translation initiation and termination seriuences. These
regulatory elements are operably linked to the sequence
to be translated. A nucleic acid sequence is operably
linked when it is placed into a functional relatir,nC~h;~
2 0 with another nucleic acid sequence . For instance, a
promoter is operably linked to a coding sequence if the
promoter affect~ its transcription or expression.
Generally, operably linked means that the DNA sequences
being linked are contiguous and, where necessary to j oin
two protein coding regions, contiguous and in reading
frame. The regulatory elements employed in the
expression vectors rrntAin;nr a polynucleotide Pnrorl;n~ a
virulence factor are functional in the host cell used for
expression .
3 o The polynucleotides of the present invention
may be prepared by any means known in the art. For
example, large amounts of the polynucleotides may be
produced by replication in a suitable host cell. ~he
natural or synthetic DNA fragments coding for a desired
3s fragment will be incorporated into recombinant nucleic
Wo 95/17~ g'~ PCr/US94/14912
acid constructs, typically DNA constructs, capable of
introduction i~to and replication in a prokaryotic or
eukaryotic cell. IJsually the DNA construct3 will be
suitable for ~-~tont us replication in a unicellular
5 host, such as yeast or bacteria, but may also be intended
for introduction to and integration within the genome of
a cultured insect , 1; ;~n, plant or other eukaryotic
cell lines. The purification of nucleic acids produced
by the methods of the present invention are described,
e.g., in Sambrook et ~L;L. (1989) or Ausubel et al. ~1987
and periodic updates ) .
The polynucleotides of the present invention
may also be produced by chemical ~ynthesis, e.g., by the
phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22:1859-1862 or the
triester method according to Matteucci et al. ~1981) ~.
Am. Chem. Soc. Oi:3185, and may be performed on
commercial automated oligonucleotide synthesizers. A
double-stranded fragment may be obtained from the single
stranded product of chemical synthesis either by
gyntht'R; 7; nr the complementary strand and annealing the
strand together under appropriate conditions or by adding
the complementary strand using DNA polymerase with an
appropriate primer ser1uence.
DNA constructs prepared for introduction into a
prokaryotic or eukaryotic host will typically comprise a
replication system recognized by the host, including the
intt~n~t~d DNA fragment encoding the desired polypeptide,
and will preferably also include transcription and
3 o translational initiation regulatory set~uences operably
linked to the polypeptide encoding segment. Expression
vectors may include, for example, an origin of
replication or autonomously replicating set~uence ~ARS)
and expression control se~lt~ncPR, a promoter, an t~nh:~nr~r
and npcp~s~ry processing information sites, such as
27
W095/17511 21 79 772 PCT/US94114912
ribosome-binding site9, RNA splice sites, polyadenylation
sites, transcriptional terminator sequences, and mRNA
- stabilizing sequences. Secretion signals from
polypeptides secreted from the host cell of choice may
5 also be included where appropriate, thus allowing the
protein to cross and/or lodge in cell membranes, and thus
attain its functional topology or be secreted from the
cell. Such vectors may be prepared by means of standard
re~:l ' ;n~nt techniques well known in the art and
10 discussed, for example, in Sambrook et ~. (1989) or
Ausubel et ~,. ~1987).
The selection of an G~ p' iate promoter and
other necessary vector sequences will be selected 80 as
to be functional in the host, and may, when appropriate,
15 include those naturally associated with mycobacterial
genes. Examples of workable combinations of cell lines
and expression vectors are described in Sambrook et al.,
1989 or Ausubel et a]., 1987); see also, e.g., Metzger et
al. 1988), Nature 334:31-36. Many useful vectors are
20 known in the art and may be obtained from such vendors as
Stratagene, New England Biolabs, Promega Biotech, and
others. Promoters such as the trp, lac and phage
promoters, tRNA promoters and glycolytic e~zyme promoters
may be used in prokaryotic hosts. Useful yeas~ promoters
25 include the promoter regions for metallothionein,
3-phosphoglycerate kinase or other glycolytic enzymes
such as enolase or glyceraldehyde-3-phosphate
dehydrogenase, enzymes responsible for maltose and
galactose ~t;1;7~t;on, and others. Suitable vectors and
30 promoters for use in yeast expression are further
described in Hitzeman et al . EP 73, 657A Appropriate
nonnative ~ n promoters might include the early and
late promoters from SV40 (Fiers et al. (1978) ~atuFe
273 :113) or promoters derived from murine moloney
35 leukemia virus, mouse mammary tumor virus, avian sarcoma
28
W095117511 ~ 9rt~ PCT/US94~14912
viruses, adenovirus II, bovine papilloma virus or
polyoma. In addition, the construct may be joined to an
amplifiable gene (e~g., DHFR) 50 that multiple copie3 of
the gene may be made. For c-~Lu~Llate f~nh~nrf~r and
other expression control sequences 3ee ~ 3o ~nhs~nrPrs and
Eukarvotic Gene Ex~ression, Cold Spring ~arbor Press,
N.Y. (1983).
While such expression vectors may r~r1; r;~t~'
on~ ,usly, they may legg preferably replicate by being
inserted into the genome of the host cell, by methods
well known in the art.
Expression and cloning vectors will likely
contain a selectable marker, a gene .-nrnA1n~ a protein
n~c~ ry f or the survival or growth of a host cell
transformed with the vector. The presence of this gene
ensures the growth of only those host cells which expres3
the inserts. Typical selection genes encode proteins
that (a) confer resistance to antibiotics or other toxic
substances, e.g. ampicillin, neomycin, methotrexate,
etc.; (b) complement auxotrophic deficiencies; or (c)
supply critical nutrients not available f~om complex
media, e.g. the gene ~nro~;n~ D-alanine racemase for
Bacilli. The choice of the proper selectable marker will
depend on the host cell, and a~ Liate markers for
dif ferent ~osts are well known in the art .
The vectors crnt~;ninrJ the nucleic acids of
interest can be transcribed in vi tro and the resulting
RNA introduced into the host cell by well known methods
(e.g., by injection. See, T. Kubo et a]., FEBS hett.
241: ll9 (1988) ), or the vectors can be i~troduced
directly into host cells by methods well known in the
art, which vary c~f~I7.'n~;nr~ on the type of cellular host,
including electroporation; transfection employing calcium
chloride, rubidium chloride calcium phosphate, DEAE-
dextran, or other substances; microprojectile
29
WO95/17511 21797,7~ PCrlUS94/~4912
!
'-- ,' t; lipofection; infection (where the vector is
an infectious agent, such as a retroviral genome); and
other methods. See generally, Sambrook et al. (1989) and
Ausubel et al. (1987). The cells into which have been
5 introduced nucleic acids described above are meant to
also include the progeny of such cells.
Large quantities of the nucleic acids and
polypeptides of the present invention may be prepared by
expressing the nucleic acids or portions thereof in
10 vectors or other expression vehicles in compatible
prokaryotic or eukaryotic host cells. The most commonly
used prokaryotic hosts are strains of Escherichia coli,
although other prokaryotes, such as Bacillus 8ubtilis or
p9~ 1nTnnAf~ may also be used.
1~ l; An or other eukaryotic hogt cells, such
as those of yeagt, fil tr7U3 fungi, plant, insect,
amphibian or avian species, may also be useful for
production of the proteins of the present invention.
Propa~atinn of mammalian cells in culture is ~er se well
20 known. See, Tissue Cultllrel Kruse and Patterson, ed.,
Academic Press (1973). Examples of commonly used
l i An host cell lines are VER0 and HeLa cells,
Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS
cell lines, although it will be appreciated by the
25 skilled practitioner that other cell lines may be
appropriate, e.g., to provide higher expression,
desirable glycosylation patterns, or other features.
Clones are selected by using markers ~l~rF.n~1in~
on the mode of the vector construction. The marker may
30 be on the same or a different DNA molecule, preferably
the same DNA molecule. The transformant may be screened
or, preferably, selected by any of the means well known
in the æt , e . g ., by resistance to such antibiotics as
ampicillin, tetracycline.
W0 9S/17511 ~ 9~ PCr/US9414912
Also included within the invention are
isolated or recombinant polynucleotides that bind to the
regions of the mycobacterial chromosome c^n1 ;1;n;~
sequences that are associated with virulence, including
antisense and triplex forming polynucleotides. As used
herein, the tarm "binding" refers to an interaction or
complexation between an oligonucleotide and a target
nucleotide sequence, ~ through 11yd~uuc:~l bonding or
other molecular forces. The term "binding~ more
specifically refers to two types of internucleotide
binding mediated through base-base hydrogen bonding. The
first type of binding is "Watson-Crick-type" binding
interactions i~ which adenine-thymine (or adenine-uracil)
and guanine-cytosine base-pairs are formed through
hydrogen bondin~ between the bases. An example of this
type of binding is the binding traditionally associated
with the DNA double helix and in RNA-DNA hybrids; this
type of binding is normally detected by hybridization
procedures .
The second type of binding is "triplex
binding". In general, triplex binding refers to any type
of base-base hydrogen bonding of a third polynucleotide
strand with a duplex DNA (or DNA-RNA hybrid) that is
already paired in a Watson-Crick manner.
The i~lvention also includes recombinant host
cells comprised of any of the above described
polynucleotides that contain a sequence ~so~ t~l with
virulence in mycobacteria, in~ l;n~ those encoding a
polypeptide, particularly a polypeptide that is
substantially homologous to the polypeptide encoded in
Figure 9, or a ~ragment thereof, or an analog thereof.
The polynucleotides of the invention may be
inserted into the host cell by any means known in the
art, including for example, transforTIlation, transduction,
and electroporation. As used herein, "recombinant host
31
W095/17511 ~1-7~7~ PCTIIJS94114912
cells", "host cells", "cells", "cell lines", "cell
cultures ", and other such terms denoting microorganisms
or higher eukaryotic cell lines cultured as unicellular
entities refer to cells which can be, or have been, used
as recipients for r.o~ ' in~nt vector or other transfer
DNA, and include the progeny of the original cell which
has been transformed. It is understood that the progeny
of a single parental cell may not n~ P~rily be
completely ;tl~ntic~l in morphology or in genomic or total
DNA complement as the original parent, due to natural,
accidental, or deliberate mutation.
~Transformation", as used herein, refers to the
insertion of an exogenous polynucleotide into a host
cell, irrespective of the method used for the insertlon,
for example, direct uptake, trAn~ -cti- ~, f-mating or
electroporation. The t:~u~nc,us polynucleotide may be
--int~;n~rl as a non-integrated vector, for example, a
plasmid, or alternatively, may be integrated into the
host cell genome.
The polynucleotides of the invention that are
essentially homologous to sequences associated with
virulence, shown in Figure 9, and in plasmids pl~HAl,
pUHA2, p~3, pUHA4, pllEIA5, pUHA6, pU~A7, pUE~Al 1 and
pr~HA16, and in plasmids pY1~3352, pYU~3353, pYU~3354 are of
use in the detection of virulent forms of mycob~ct~ria in
biological samples. As used herein, a "biological
sample~ refers to a sample of tissue or fluid isolated
from an individual, including but not limited to, for
example, plasma, serum, spinal fluid, lymph fluid, the
~rt~rn~l sections of the skin, respiratory, intestinal,
and genitourinary tracts, tears, saliva, milk, blood
cells, tumors, organs, and also samples of ~.n vitro cell
culture constituents ~including but not limited to
~ n~l;tjr~n~d medium resulting from the growth of cells in
32
WOssrl7sll 2'~9i`~2' PCTrUS94rl4912
cell culture medium, putatively virally in~ected cells,
r~ ' jn:qnt cells, and cell rr~r~nQnt~
rrsing the disclosed portions of the isolated
polynucleotides associated with virulence as a basis,
5 oligomers of approximately 8 nucleotides or more can be
prepared, either by ~Yr; ~1 rn rom recombinant
polvnucleotides or synthetically, which hybridize with
the mycobacterial 3equences in the r~ and are
useful in identification of myrrharto~ia with the
lO virulence associated trait. The probes for
pol,vnucleotides associated with virulence are a length
which allows the detection of the virulence associated
se¢uences by hybridization. ~rhile 6-8 nucleotides may be
a workable length, serluences of 10-12 nucleotides are
15 preferred, and at least about 20 nucleotides appears
optimal. These probes can be prepared using routine
methods, including automated oligonucleotide synthetic
methods. For use as probes, complete complementarity is
desirable, though it may be l~nn~c~ ry as the length of
20 the fragment is increased.
For use of such probes as diagnostics, the bio-
- logical sample to be analyzed, such as blood or serum,
may be treated, if desired, to extract the nucleic acids
rrnt~inl-~l therein. The resulting nucleic acid from the
sample may be subjected to gel electrophoresis or other
size separation techniques; alternatively, the nucleic
acid sample may be dot blotted without size separation.
The probes are usually labeled. Suitable labels, and
methods for l~h~11nJr probes are known in the art, and
include, ~or exampIe, radioactive labels incorporated by
nick translation or kinasing, biotin, fluorescent probes,
and chemiluminescent probes. The nucleic acids extracted
f rom the sample are then treated with the labeled probe
under hybridization conditions of suitable stringencie~.
The probes can be made completely complementary
Wo 95/17511 1 ~7 77,? PCrlUS94/14912
to the virulence encoding polynucleotide. Therefore,
usually high stringency conditions are desirable in order
to prevent false positives. The stringency of
hybridization is determined by a number of factors during
5 hybridization and during the washing procedure, including
temperature, ionic strength, length of time, and
rrn, ~ntration of formamide. Thege factorg are outlined
in, for example, Maniatis, T. (1982).
It may be desirable to use amplif ication
lO techniques in hybridization assays. Such techniques are
known in the art and include, for example, the polymerase
chain reaction (PCR) technique described which is by
Saiki et al. (1986), by Mullis, U.S. Patent No.
4,683,195, and by Mullis et al. U.S. Patent No.
4, 683, 202 .
The probes can be packaged into diagnostic
kits. Diagnostic kits include the probe DNA, which may
be labeled; alternatively, the probe DNA may be unlabeled
and the ingredients for l;lh~l ;n~ may be inrl~ in the
kit in separate cont~;n~rs. The kit may also contain
other suitably packaged reagents and materials needed for
the particular hybridization protocol, for example,
standards, as well as instructions for conducting the
test .
~olypeptides encoded within the sequences
associated with virulence, and fragments and analogs
thereof are also included as ~mho~ of the
invention. The polypeptide encoded in the large ORF in
Figure 9 is a putative sigma factor; thus, the intact
3~ polypeptide may exhibit the following biological
activities: (l) binding to mycobacterial core RNA
polymerase, (b) activation of promoter recognition;, and
may include (c) DNA melting and (d) inhibition of
nonspecific transcription. Methods to determine these
biological functions are known in the art, and for
34
WosS/17511 ~,~l9~rl PCT/IJS94114912
example are reviewed in J.D. Helmann and M.J. Chamberlin,
Ann. Rev. ;3iochem. (1988) 57, 839-872. Also ;nrl~ fi as
a biological activity of any specific polypeptide is the
binding of the polypeptide to an antibody that is
5 directed to one or more epitopes on that polypeptide.
The invention include3 polypeptides and analogs or
~ragments thereof that are essentially homologous to the
polypeptide encoded in the large ORF in ~igure 9, and
exhibit at least one of the biological activities
lO associated with sigma factor, or alternatively, inhibits
at least one oi the biological activities associated with
sigma factor.
The term ~polypeptide~ refers to a polymer of
amino acids and does not refer to a specific length of
15 the product; thus, peptides, oligopeptides, and proteins
are; n~ within the definition of polypeptide . This
term also doec not refer to or exclude post-expression
mod;f;r~ nR of the polypeptide, for example,
glycosylations, acetylations, rhnsph~rylations and the
20 like. Included within the de~inition are, for example,
polypeptides crnt~;n;n~ one or more analogs of an amino
acid ~inr~ in~, for example, unnatural amino acids,
e~c. ), polypeptides with substituted linkages , as well as
the modifications known in the art, both naturally
25 occurring and non-naturally occurring.
Ordinarily, the polypeptides of the present
invention will be at least about 509~ homologous to the
polypeptide encoded in the large ORF o~ ~igure 9,
designated herein as ~virulence asgociated sigma factor
30 l" ~also referred to herein as "rpoV"~, preferably in
excess of about 90~, and, more preferably, at least about
95~ homologous. Also included are proteins encoded by
DNA which hybridize under high or low stringency
condition~, to nucleic acids encoding virulence
35 associated sigma factor l, as well as closely related
~1~
Wo 95/17511 ;~? PCr/US94/14912
polypeptides or proteins retrieved by anti3era to
virulence associated sigma factor 1.
The length of polypeptide sequences compared
for homology will generally be at least about 16 amino
5 acids, usually at least about 20 residues, more usually
at least about 24 residues, typically at least about 2
residues, and preferably more than about 35 residues.
The term "substantial homology~ or "substantial
identity", when referring to polypeptides, indicates that
10 the polypeptide or protein in question exhibits at least
about 30~ identity wit~ an entire naturally occurring
protein or a portion thereof, usually at least about 709
identity, and preferably at least about 95~ identity.
Homology, for polypeptides, is typically
15 measured using sequence analysis software. See, e.g.,
Sequence Analysis Software Package of the Genetics
Computer Group, University of Wisconsin Biotechnology
Center, 1710 University Avenue, Madison, Wisconsin 53705.
Protein analysis software matches similar sequences using
20 measure of homology assigned to various substitutions,
deletions, substitutions, and other modif ications .
Conservative substitutions typically include
substitutions within the following groups: glycine,
alanine; valine, isoleucine, leucine; aspartic acid,
25 glutamic acid; asparagine, glutamine; serine, threonine;
lysine, arginine; and phenyl~1Antnp~ tyrosine.
A polypeptide "fragment, " "portion, " or
" segment " is a stretch of amino acid residues of at least
about 5 amino acids, of ten at least about 7 amino acids,
30 typically at least about 9 to 13 amino acids, and, in
various ~ c, at least about 17 or more amino
acids .
The terms "isolated," "substAnt;~lly pure," and
"subst~ntiAlly homogenous" are used interrhAn~PAhly to
3 5 describe a protein or polypeptide which has been
36
Wo 95/17511 ~ Pcrluss4llJs~2
separated from components which naturally ac~ _- y it.
A monomeric pxotein i8 sUbStAnti;-lly pure when at least
about 60 to 75% of a sample exhibits a single polypeptide
sequence. A subst~nti~lly pure protein will typically
5 comprise about ~o to go~ W/W of a protein sample, more
usually about 95$, and preferably will be over about 999~
pure. Protein purity or ~ ,~..eity may be indicated by
a number of means well known in the art, such as
polyacrylamide gel electrophoresis of a protein sample,
lO followed by visualizing a single polypeptide band upon
staining the gel. For certain purposes higher resolution
can be pxovided by using HP~C or other means well known
in the art.
A protein is considered to be isolated when it
15 i3 separated from the rorltAminAntf~ which Arrr~Any it in
its natural state. Thus, a polypeptide which is
chemically synthesized or syl~th~ d in a r.olllllAr
system different from the cell from which it naturally
originates will be substAnti~lly free from its naturally
2 0 assoc iated, - t ~ .
The present invention provides polypeptides
which may be purified from mycobacteria as well as from
other types of cells transformed with recombinant nucleic
acids encoding these proteins . Such protein purif ication
25 can be AC. ~ hl~d by various methods well known in the
art, and include those fl~rr;h~d, e.g., in Guide to
Protein Purificationr ed. M. Deutscher, vol. 1~2 of
Method8 in Rn7Ymolorv (Academic Press, Inc.: San Diego,
l990) and R. Scopes, Pxotein Purification: Prinçi~les and
3 0 Practice, Springer-Verlag: New York, 1982 .
If n~rr~sAry, the amino acid sequence of the
proteins of the present invention can be fl~t~rmir.o~ by
pxotein sequencing methods well known in the art.
The present invention also provides f or
35 polypeptides or fragments thereof which are sub8t~ntiA1 ly
37
Wo 95117511 1 ;~9 7~72 PCr/US94/14912
homologous to the primary structural sequence of the
virulence associated sigma factor l (also called r,ooVJ.
The present invention also embraces ~n YiVo or 1 Yitro
chemical and bio~hPmi rAl modifications that incorporate
5 unusual amino acids. Such modifications include, for
example, acetylation, carboxylation, ~h~spht~rylation~
glycosylation, ubiquitination, labelling, e.g., with
ri9rl;( nll--l ;r1F'R, various enzymatic modi~ications, as will
be readily appreciated by those well skilled in the art.
10 A variety of methods for lAhF-l l; ng polypeptides and of
substituents or labels useful for such purpose_ are well
known in the art and include r~ t i ve isotopes such as
32p, ligands, which bind to labeled antiligands (e.g.,
ant ibodies ), f luorophores , chemi luminescent agents ,
15 enzymes, and antiligands which can serve as specific
binding pair members for a labeled ligand. The choice of
label depends on the sensitivity required, ease of
conjugation with the primer, stability requirements, and
available in~LL, ~tion. Methods of lAh-~llin~
20 polypeptides are well known in the art. See, e.g.,
Molecular Cloninq: A LaboratorY Manual, ~ ed., Vol. l-
3, ed. Sambrook, et ~., Cold Spring EIarbor Laboratory
Press (1989) or Current Protocols in Molecular Biolo~v,
ed. F. Ausubel et al., Greene p11hl;ah;n~ and Wiley-
25 Interscience: New York (1987 and periodic updates).
~ 3esides subst~nt;~lly full-length polypeptides,
the present invention provides for fL _ 8 of the
polypeptides capable of binding to ~ntihQfi;es directed to
virulence associated sigma factor l. As used herein, the
30 term fragment or segment, as applied to a polypeptide,
will ordinarily be at least about 5 to 7 contiguous amino
acids, typically at least about 9 to 13 contiguous amino
acids, and most preferably at least about 20 to 30 or
more contiguous amino acids.
W095117511 ~ 9'lrl?' PCrlUS94/14912
The present invention al60 provides for fusion
polypeptides comprising the virulence associated sigma
factor 1 or fragments thereof. Homologous polypeptides
may be fusions between two or more sequences derived from
5 the virulence associated sigma factor 1 or between the
sequences of the virulence A~ROCi ~ecl protein and a
related protein. Likewise, heterologous fusions may be
constructed which would exhibit a combination of
properties or activities of the derivative proteins.
See, e.g., Godowski et al. (1988) ~S~.e~ce 241:812-816.
E'usion proteins will typically be made by
recombinant nucleic acid methods, but may be chemically
synthesized. Techniques for synthesis of polypeptides
are described, for example, in Merrifield (1963) ~. Amer.
Chem. Soc . 85 :2149-2156.
The polypeptides of the present invention may
be used in the preparation of vaccines to treat and/or
prevent diseases associated with mycobacterial
infections. "Treatment" as used herein refers to
prophylaxis and/or therapy.
The polypeptides can be prepared as discrete
entities or incorporated into a larger polypeptide, and
may f ind use as described herein. The immunogenicity of
the epitopes of the polypeptides of the invention may
also be f~nh~nced by preparing them in ~ n or yeast
systems fused with or assembled with particle-forming
proteins such as, for example, that associated with
hepatitis s surf ace antigen . See , e . g ., ~. S . Pat . No .
4,722,840. Vaccines may be prepared from one or more im-
-, 1 c polypeptides derived from virulence associated
polypeptides, and more particularly from virulence
associated sigma factor 1.
The preparation of vaccinea which contain an
immunogenic polypeptide~s) as active ingredients, is
known to one skilled in the art. Typically, such
39
Wos5/17511 17~ PCT/US94/14912
vaccines are prepared as injectables, either as liquid
solutions or suspen9ions; solid form.s suitable for solu-
tion in, or suspension in, liquid prior to injection may
also be prepared. The preparation may also be
5 emulsified, or the protein encapsulated in liposomes.
The active immunogenic ingredients are often mixed with
PYA;ripntR which are pharm.aceutically acceptable and
_-tihle with the active ingredient. Suitable
excipients are, for example, water, saline, dextrose,
lO glycerol, ethanol, or the like and combinations thereof.
In addition, if desired, the vaccine may contain minor
amounts of auxiliary substances such as wetting or
emulsifying agents, pH buffering agents, and/or adjuvants
which enhance the ef f ectiveness of the vaccine . Examples
15 of adjuvants which may be effective include but are not
limited to~ m;n1~m hydroxide,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,
referred to as nor-MDP),
20 N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (l' -
2 ' -dipalmitoyl-sn-glycero-3 -hydroxyphosphoryloxy) -ethylam
ine (CGP 19835A, referred to as MTP-PE), and RIBI, which
-~-,nt_;nA three, ~ ~~tr extracted from bacteria,
~-,nnph~-,5ph-ryl lipid A, trehalose dimycolate and cell
2~ wall skeleton (MPL+TDM+CWS) in a 29~ squalene/~ween 80
,n. The effectiveness of an adjuvant may be
detPrm; nPd by measuring the amount of antibodies directed
against an immunogenic polypeptide c~-,nt_;n;n~ an ~poV
antigenic sequence resulting from administration of this
3 0 polypeptide in vaccines which are also comprised of the
various adj UV~Ilts .
The vaccines are convPnt; -~n~l ly administered
parenterally, by injection, for example, either
subc-1t~nP~ 1c1 y or intr-m~-c~ rly . Additional
3~ formulations which are suitable for other modes of
W09S/17511 ~ ~ ~7~7~ PCrl~,7S94/14912
administration include suppositories and, in some cases,
oral formulations or formlllAtinnc7 suita}~le for
distribution as aerosols. For auppositories, traditional
binders and carriers may include, f or eXample,
5 polyalkylene glycols or triglycerides; such auppositories
may be formed from mixtures n ^nt~7;nin~ the active
ingredient in the range of 0.59~ to 10~, preferably 1~-2~.
Oral form~ll A7; nnR include such normally employed
excipients as, for example, pharmaceutical grades of
10 mannitol, lactose, starch, magnesium atearate, sodium
saccharine, cellulose, magnesium ~ArhnnAte~ and the like.
These compositions take the form of Anll~ti~mc7, su3pen-
sions, tablets, pills, capsules, sustained release
formulations or powders and contain 10~-95~ of active
ingredient, preferably 259;-709~.
The proteins may be formulated into the vaccine
as neutral or salt forms . Pharm--e7~ Al 1 y acceptable
salts include the acid addition salts (formed with free
amino groups of the peptide~ and which are formed with
20 inorganic acids such as, for example, hydrochloric or
rhnsphnric acidg, or with organic acids such as acetic,
oxalic, tartaric, maleic, and the like. Salts formed
with the free carboxyl groups may also be derived from
inorganic baaes such as, for example, sodium, potassium,
25 ammonium, calcium, or ferric hydroxides, and such organic
bases as isopropylamine, trimethylamine, 2-ethylamino
ethanol, histidine, procaine, and the li,ce.
The vaccines are administered in a manner
_atihle with the dosage formulation, and in such
30 amount as will be prophylactically and/or therapeutically
effective. The quantity to be administered, which is
generally in the range of 5 mi.LUyL~.I.,O to 250 mi.:,u~7,cLI,~
of antigen per dose, depe~is on the subject to be
treated, capacity of the subject's immune system to
35 8ynth~Ai7.o antibodies, and the degree of protection
41
Wo 95/17511 ~ 3 7 pCrraS94/14912
i7æ
desired. Precise amounts of active ingredient required
to be administered may depend on the judgment of the
practitioner and may be peculiar to each subject.
T~e vaccine may be given in a single dose
5 schedule, or preferably in a multiple dose srhod1ll e . A
multiple dose schedule iB one in which a primary course
of vAcn;nAtinn may be with l-lO separate doses, followed
by other doses given at subsequent time intervals
required to maintain and or reenforce the immune
lO response, for example, at 1-4 months for a second dose,
and if needed, a subsequent dose (8) after several months .
The dosage regimen will also, at least in part, be
determined by the need of the individual and be dependent
upon the judgment of the practitioner.
15 ~ In addition, the vaccine ~nntAin;n~ the im-
munogenic mycobacterial antigen ~8) may be administered in
conjunction with other immunoregulatory agents, for ~ =
example, immune ~lnhlll inq, as well as antibiotics.
The immunogenic virulence associated antigens
2C may be used for the preparation of Ant;hn~;oc The
immunogenic polypeptides prepared as described above are
used to produce Ant; ho~ c, including polyclonal and
nnrlnnAl If polyclonal AntihnriiF~c a~e desired, a
selected mammal (e.g., mouse, rabbit, goat, horse, etc.)
25 is i ; 7~ with an; ngon; c polypeptide bearing an
~poV epitope(s) Serum from the; i7ed animal is
collected and treated according to known procedures. If
serum ~-nntA;n;ng polyclonal antibodies to an ~poV epitope
~ontA;nc Ant;ho~;es to other antigens, the polyclonal
30 Ant;hn~ie5 can be purified by i --ffinity
chromatography. Techniques for producing and processing
polyclonal antisera are known in the art, see for
example, Mayer and Walker (1937).
~nnnt~l nnAl antibodies directed against ~poV
35 epitopes can also be readily produced by one skilled in
42
W095/17511 ~ 9,~ PCr/US94114912
the art. The ~eneral methodology for making monoclonal
antibodies by hybridomas is well known. Immortal
antibody-producing cell lines can be created by cell fu-
sion, and also by other technigues such as direct tran3-
5 ormation of B lymphocytes with oncogenic DNA, ortran~fection with Epstein-Barr virus. See, e.g., M.
Schreier et al. (1980); Hammerling et al. (1981); Kennett
et al. (1980); see ~, U.S. Patent Nos. 4,341,761;
4,399,121; 4,427,783; 4,444,887; 4,466,917; 4,472,500;
4, 4gl, 632; and 4, 493, 890 . Panels of monoclonal
ihn~;es produced ayaingt rpoV epitopeg can be screened
for various properties; i.e., for isotype, epitope affin-
ity, etc.
Antibodies, both monoclonal and polyclonal,
15 which are directed against zpoV epitopes are particularly
useful in rl1A~nnsic, and those which are neutralizing may
be useful i~ passive immunotherapy. Monoclonal
antibodies, in particular, may be used to raise
anti-idiotype :~ntihnrli,..~ Anti-idiotype antibodies are
immunoglobulins which carry an "intf~rn~l image" of the
antiyen of the infectious agent against which protection
is desired. See, for example, Nisonoff, A., et al.
(19811 and Dreeaman et al. (1985) . I'echnigues for
raising anti-idiotype ~n~iho~ies are known in the art.
See, for examp~e, Grzych (1985), MacNamara et al. (1984),
and Uytdehaag et al. (1985). These anti-idiotype
Antihn~ieg may algo be useful for treatment, vaccination
and/or diagnosis of mycobacterial inf ections, as well as
for an elucidation of the immunogenic regions of rpoV
3 0 antigens .
Both the virulence associated polypeptides and
~ntiho~lieg to them are useful in ;r~ nn~csays to detect
presence of antibodies to mycobacteria, or the presence
of the virulence associated antigens, and particularly
the presence of virulence ~csn~ ted rpoV in biological
43
Wo9~117511 17977~ PCI~/US94/14912
samples. Design of the ~ Rsays is subject to a
great deal o variation, and many formats are known in
the art . The ; - - RRAy will utilize at least one
epitope derived from a virulence associated polypeptide,
and particularly virulence associated rpoV. In one
'-'ir ~, the; -ARgay uses a ;nAtion of
~r; ~Op~R derived from the virulence associated
polypeptide. These epitopes may be derived irom the same
or from different bacterial polypeptides, and may be in
separate recombinant or natural polypeptides, or together
in the same recombinant polypeptides. An; Inr~ARRAy may
use, for example, a monoclonal antibody directed towards
a virulence associated polypeptide epitope (s), a combina-
tion of monoclonal Antiho~;es directed towards epitopes
of one mycobacterial antigen, monoclonal ~nt; ho~ R
directed towards epitopes of different mycobacterial
antigens, polyclonal Ant; hoA i e8 directed towards the 8ame
antigen, or polyclonal Ant;hori;,~R directed towards
different antigens. Protocols may be based, for
example, upon competition, or direct reaction, or
sandwich type assays. Protocols may also, for example,
use solid supports, or may be by immunoprecipitation.
Most assays involve the use of labeled antibody or
polypeptide; the labels may be, for example, enzymatic,
fluorescent, chemiluminescent, radioactive, or dye
molecules. Assays which amplify the signals from the
probe are also known; examples of which are assays which
utilize biotin and avidin, and enzyme-labeled and
mediated; Inl~Accays~ such as ELISA assays.
Typically, an I ~ARRAy for an antibody(s) to
a virulence associated polypeptide, and particularly to
virulence associated zpoV will involve selecting and
preparing the test sample suspected of containi~g the
antibodies, such as a biological sample, then incubating
it with an antigenic (i.e., epitope-containing) virulence
44
Wo 95/17511 2 1 ~ ~ ~ 7 2 PCT/US9411~912 ~
assoclated polypeptide (8) under conditiong that allow
antigen-antibody complexes to form, and then ~f~tPctin~
the formation of such complexes. Suitable incubation
conditions are well known in the art. The immunoas3ay
may be, without limitations, in a heterogenous or in a
homogeneoug fQrmat, and of a standard or competitive
type .
In a heterogeneous format, the polypeptide is
typically bound to a solid support to facilitate separa-
tion of the sample from the polypeptide after inmlh~tinn
Examples of solid supports that can be used are nitro-
cellulose (e.g., in membrane or microtiter well form~,
polyvinyl chloride (e.g., in sheets or microtiter wells~,
polystyrene latex (e.g., in beads or microtiter plates,
polyvinylidine fluoride (known as Immulon), diazotized
paper, nylon ' ~ es~ activated beads, and Protein A
beads. For example, Dynatech Immulon 1 or Immulon 2
microtiter plate3 or 0 . 25 inch polystyrene beads (Preci-
sion Plastic Ball) can be used in the heterogeneous
format. The solid support ~ nntAin~n~ the antigenic
polypeptide is typically washed after separating it from
~he test sample, and prior to detection of bound antibod-
ies. Both standard and competitive format8 are known in
the art.
Complexes formed comprising anti-rpoV antibody
(or, in the case of competitive assays, the amount of
competing antibody) are detected by any of a number of
known te~ hni~ ren~lin~ on the format. For example,
llnl ;IhPl Pd anti-virulence associated polypeptide
~ntihorlies in the complex may be detected using a
conjugate of ~nt;~rPnn~eneic Ig complexed with a label,
(e.g., an enzyme label).
In i o~ays where the virulence associated
polypeptides are the analyte, the test sample, typically
a biological sample, is incubated with antibodies
~5
Wo 95/17511 21 79 7 72 ~S94114912
directed against the virulence associated polypeptide
under conditions that allow the formation of
antigen-antibody complexes. It may be desirable to treat
the biological sample to release putative bacterial
5 ~ nt~ prior to testing. Various formats can be
employed . Por example, a " sandwich assay~ may be
employed, where antibody bound to a solid support i9
incubated with the test sample; wa3hed; incm~h~ted with a
second, labeled antibody to the analyte, and the support
10 is washed again. Analyte is detected by det~rminin~ if
the second antibody is bound to the support. In a
competitive f ormat, which can be either heterogeneous or
homogeneous, a test sample is usually inr~lh~t,~d with
antibody and a labeled, competing antigen i8 also
l~ incubated, either se~nt;~lly or simultaneougly. These
and other formats are well known in the art.
Also included as an ~ i r ~ of the invention
is an; ~AI~.Cay kit comprised of one or more
polypeptides of the invention, or antibodies to a
20 polypeptide associated with virulence, and a buffer,
packaged in suitable e~nt~in.or8.
In addition,, , ~ul~ds which block the activity
of virulence factor associated polypeptides and
particularly virulence ~so~ i Rted rpoV, may be prepared
2~ llt;li7in~ the gequence information of provided herein.
This is performed by ~vc:~ x~Lessing the polypeptide,
purifying the polypeptide, and then performing X-ray
crystallography on the purified virulence associated
polypeptide to obtain its molecular structure. Next,
3 0 ~ ,~uullds are created which have similar molecular
structures to all or portions of the polypeptide or its
substrate. The ~ , iuulds are then combined with the
polypeptide and attached thereto 80 as to block one or
more of its biological activities.
3~
46
Wo 95117SII ` ~ ~ ~ PCrllJS94/14912
The polynucleotides of the invention may also
be used to produce or improve live attenuated or killed
tuberculosis vaccines. For example a vaccine strain may
be produced by mutating a virulence associated
5 polynucleotide, and particularly one encoding virulence
asaociated sigma factor 1. The mutated strain may then
be formulated into a vaccine and administered to treat
mycobacterial in~ections. In addition, virulence
associated polynucleotides may be added to BCG vaccine
lo strains to provide attenuated mutant tuberculosis
vaccines .
The invention also f~nrnnlm:~cses a new approach
f or determining f actors associated with virulence or
other properties of interest in other genera of bacteria
15 by showing that an arginine to histidine change near the
C-terminal end of a principal sigma factor, and in
particular at the equivalent site to that which occurs in
M. bovis AtCC35721, is not lethal but causes an
alteration in the specificity of promotion of the sigma
2 o ~actor . Such a change could be engineered in the
principal sigma factor in species o other genera of
bacteria using techniques known in the art, including for
example, site directed mll~p~nP~i ~ and homologous
r~l ;n:lt jnn, Identification and subsequent
25 investigation of the genes whose promotion is altered by
such a change could be performed using techniques known
to one of skill in the art, for example, comparative
protein electrophoresis, partial protein sequencing and
reverse genetic methods. One might also use, for
30 example, in vivo methods for identifying the level of
promotion of different promoters in the presence of
normal and altered sigma factors. The results of these
studies should reveal genes whose promotion changes
significantly when promoted by an altered principal sigma
35 factor. Such genes may be potential targets for new
47
WO95117511 21 79772 PCTIIIS94114912
drugs or they could be targets for inactivation to
generate new strains for use in vaccines or strains with
other desirable properties.
The following examples are provided only for
illustrative purposes, and not to limit the 6cope of the
present invention. In light of the present disclosure
numerous ' ~i s within the scope of the claims will
be apparent to those of ordinary skill in the art.
Exam~le
Is~rl~TIoN OF A vTRrrr,rzNc~ FACTOR OF MYCOBACT~RT~ USING A
GUIl~ PIG COMPLEM~TATION ASSAY.
Virulent tuberculosis complex strains were
cultured as described previously (Collins and de Lisle
1984). Mycobacterial species were identified by standard
methods .
For preparation of genomic DNA, tuberculosis
complex strains were grown on standard mycobacterial
media, harvested into buffer and inactivated by heating.
Genomic DNA was prepared f orm the organisms and
partially digested with a range of c~m~ nt~ations of
Sau3AI. Fragments of 30-50 kb from these digestions were
prepared using sucrose gradient centrifugation and
ligated to l~clI-digested pYU!3l78 DNA that had been
treated with calf intestinal phosphatase. The ligation
mixture was in vi tro-packaged into ~ phage heads and
transduced into E~cherichia coli. The kanamycin
resistant recombinant clones were pooled and cosmid DNA
3 0 was prepared using standard plasmid isolation methods .
The variability of members of the library was
es~hl i ~ d.
A tuberculosis complex strain of lowered
virulence for guinea pigs (referred to subsequently as
avirulent) was cultured in roller bottles and organisms
48
Wo 95117511 PCTIUS94114912
2~
were prepared and electroporated with a library of
pYUB178::virulent-tuberculosis-complex-DNA. The
electroporated organisms were plated onto media
containing kanamycin and kanamycin resistant clones were
5 pooled to form a library. Each member of this library
had the C1LL~ of the avirulent tuberculosis organism
into which a cosmid with an insert of genomic DNA from a
virulent tuberculosis complex strain was integrated. The
library was cultured in liquid media and aliquots were
10 in~clllAtl~cl into guinea pigs. Separate guinea pigs were
also inoculated with the matching avirulent tuberculogis
complex strain as a control. The most clear cut
distinction between virulent and avirulent strains was in
the presence or absence of gross lesions in the spleen.
The method f or vinllence testing in guinea pigs
was adapted f rom the procedures described in the Trudeau
Mycobacterial Culture Collection catalogue, (Anon, 1972) .
Albino, outbred guinea pig8 were inoculated
sllhc~lt~n~ously in the flank. Libraries and individual
20 strains of mycobacteria were inoculated into at least
three guinea pigs which were kept in filtered-air,
v~nt i 1 ~t---l animal cages . A~imals were sacriiced
approximately 6 and 13 weeks after inor~ ti~n and
~T:~min~rl for the presence of gross lesions of
25 tuberculosis. Samples from the injection site, the
prefemoral lymph nodes and spleen were cultured for
mys-ohE~ t~ ia using previously described methods.
Formalin-fixed tissue3, from the spleen, liver, kidney
and lung were embedded in parafin, sectinn~d at 3-5 ~Lm,
30 and stained with either hematoxylin and eosin ~XE) or by
the Ziehl-Neelsen method.
A. Vinll ,~nt T~herc~l osiR Strain Used to ~ke
LibrarY
A virulent M. bOV~8 strain was isolated from
35 bovine tissue submitted to the Wallaceville Animal
49
WO 95117511 79 772 PCr/lJS94/14912
Research Centre, Jpper Hutt, New Zealand. The strain,
isolated ~rom bovine tissue with the acceqsion number
89/5276, was designated WAg200 and was cultured as
described previously (Collins and de ~isle 1984). The
strain was also shown to be virulent for guinea pigs.
Bacteriological itl~ont; ~i cation of the strain as M. bovis
was based on colony morphology, slow growth, acid-fast
staining, susceptibility to thiophene-2-carboxylic acid
hydrazide and isoniazid, and growth on pyruvate-
supplemented but not glycerol-supplemented media. The
strain was also characterized by restriction f ragment
analysis (Collins et al. 1993) . In infected animal
experiments described below, bacteriological
identification of reisolated M. bovls strains was based
on colony morphology, slow growth and growth on pyruvate-
supplemented media.
B. DNA Pre~aratiQr~
M. bovls WAg200 was cultured under biosafety
r~r~n~;l; t at 37C on 40 x 85 mm petri dishes of 7Hll
Middlebrook (Difco) media containing oleic acid, albumin,
dextrose, serum, lysed red blood cells, o.o59,~
polyoxyethylene sorbitan monooleate (Tween-80) and
pyruvate (G~ h~r and Horwill 1977) . The organisms
were harvested into 7 Falcon tubes each ~7nt;~inin~ 50 ml
phosphate buffered saline (0.14 M NaCl, 4 mM KCl, 8 mM
Na2HPO4, 2 mM KH2P04; p~ 6 . 5 ) and inactivated by heating
at 75C for 35 min. After centrifugation, the yield in
each tube was 1-1. 5 g wet weight organisms . Genomic DNA
was prepared from the organisms using a scaled up version
of the method described by van Soolingen et al. (1991).
The total yield of DNA af ter extraction of all organisms
was 3 0 ~ ~lg in 1 ml .
WO95/17511 ~9~ PCr/US94114912 ~
C. B. coli co3mid ~ ;hrary of k~. bo~ W~ oo
M. bovis WAg200 DNA was partially digested with
a range of rnn~pntrations of Sau3AI and digestions having
the largest yield o~ 30-50 kb fL _ tq were selected
after analytical electrophoresis on 0.4~ agarose gels
(Jacob3 et al. 1991). ~L _ tr: of 30-50 kb from these
digestions were ~Le~clLed using sucrose gradient
centrifugation (Weis 1991) and ligated to BclI-digested
pY~3178 DNA that had been treated with calf intestinal
phosphatase. The final genomic DNA rnnrpntration in the
10 ~Ll ligation mixture was 200 ng/~l and the DNA molar
ratio of insert to vector was 1: 20 . Four 1ll of the
ligation mixture was in vitro-packaged with the GigaPack
II Gold Packaging Extract ~Stratagene, La Jolla, CA)
according to the manufacturer's procedure. The in vitro-
packaged lysate was tr~n~ rPd, using previously
described methods (.Jacobs et al. 1991), into B. coli.
The kanamycin resistant rern~i n~lnt clones were pooled
and inoculated ~nto LB broth cnntAin;nrJ 25 ~g/ml
kanamycin. Cosmid D~A was prepared using standard
plasmid isolation methods of alkaline lysis and cesium
chloride gradient centrifugation (Sambrook et al. 1989).
Aliquots of the library were stored frozen at -70C and
cosmid DNA preparations were stored at -20~C.
The total number of re~ i n;~nt B. coli clones
produced was apprnYir~tPly 20,000. These clones were
pooled and the library of pY~317~: :M. bovis WAg200
cosmids was amplified as a plasmid prepdration. This
~re~dL~tion was_performed by culturing the pOolêd clones
in 750 ml Ls media rnnt:linln~ 25 ,ug/ml kanamycin. Both
before and after the plasmid amplification of the
library, cosmids from 20 randomly selected clones were
shown to have dif ferent restriction patterns .
Plasmids and M. bovis strains used in this
study are listed in Tables 1 and 2.
5i
Wo 95117SII ,~97~ PCT/US94/14912
TA~3LE 1
M. bovis strains used in this study
M . boYi 8
strain or
clone Description Source
ATCC35721 Low virulence strain ATCC
WAg200 Virulent strain isolated in N.Z. G. de I,isle
WAg300 ATCC35721 rnntAin;n~ pUHAl This study
WAg301 ATCC35721 rnntAin;n~ pU~A3 This study
WAg302 ATCC35721 ~nnt~;n;n~ pUHA4 This study
WAg303 ATCC35721 rnntA;nin~ pUHA5 This study
(Junction Fragment Pattern 1)
WAg304 ATCC35721 rontA;n;n~ pUHA5 This study
~Junction Fragment Pattern 2)
WAg305 ATCC35721 rnnt::l;n;n~ pUHA5 This study
(Junction Fragment Pattern 3)
WAg306 ATCC35721 cnntA;n;n~ pl~lA6 This study
tJunction FL__ -t Pattern 1)
WAg307 ATCC35721 rnnt~;n;n~ pUHA6 This study
(Junction Fragment Patter~ 2)
WAg308 ATCC35721 ~-nrtA;n;n~ pUHA6 This study
(Junction FL_._ ' Pattern 3)
WAg309 ATCC35721 cnntA;n;n~ p~lHA7 This study
(Junction Fragment Pattern 1)
WAg310 ATCC35721 rnnt~;n;n~ pUE~A7 This study
(Junction Fragment Pattern 2)
WAg311 ATCC35721 ~-nntAinin~ p~HA7 This study
l~tic~n F:~ t ~attern 3)
WO 95/17511 2 ~ 7 9 ~ ~ 2 - PCr/US94114912 1~
WAg320 ATCC35721 cnnt~;n;n~ 3 kb This study
fragment ofi WAg200 that restores
virulence
TAl8LE 2
Plasmids used in this study
Plasmid Description Source
pYU~3178 Integrating cosmid shuttle W. .Jacobs
vector
pUHAl pYUB178: :WAg200 cosmid which This study
restores virulence to ATCC35721
pl~HA2 pYUB178 cnntA;n;ng 6 kb oi- This study
pUHAl insert
p~3HA3 pYUB178: :WAg200 cosmid This study
overlapping pUHA2
pT~HA4 pYU1~178: :WAg200 cosmid This study
overlapping pUElA2
pUHA5 pY1~3178: :WAg200 cosmid This study
overlapping pUHA2
pUHA6 pY~3178: :WAg200 cosmid This study
overlapping pU~A2
pUHA7 pYUB178: :WAg200 cosmid This study
overlapping p~3HA2
pUHA8 pYUB178 with PacI sites on both This study
sides oi- the BclI cloning site
pUHA9 pBluescript II KS (+) b~ith PacI This study
sites on both sides o~ the BclI
site
53
WO 95/17511 1 7~ 77~ PCr/US94/14912
pUHAll pUHAg containing 3 kb fragment This study
from WAg320
pUHA16 pUHAll with 3 kb fragment in This study
reverse ori~ont~t;nn
D. Tran8formation of cosmid librarY into avirulerlt 1~.
bovi 8
The receptor strain used was M. bovis ATCC35721
10 which had lowered virulence for guinea pigs. For
simplicity this strain i8 subsequently reerred to as
avirulent. It was ino~ulated into 2 x 100 ml Middlebrook
7H9 broth ~Difco) cnnt~in;n~ albumin, glucose, glycerol
and Tween-80 as described (Jacobs et al. 1991~. The
15 cultures were grown in roller bottles at l revolution/min
to an O,D. at 600nm of 0.18. The organisms were washed
and cnnc~ntrated to a volume of 1 ml in cold 109~ glycerol
and 0 .4 ml were electroporated with 4 ~Ll of pYU~3178: :M.
bovis WAg200 cosmid library DNA (1 ~g/~Ll) as described by
20 Jacobs et al. (1991). After electroporation, the
organisms were cultured at 3 7 C on the same media used
for DNA preparation but without the addition of oleic
acid, serum or lysed red blood cells and with the
addition of 19~ sodium pyruvate and 10 ~Lg/ml kanamycin.
Approximately 4000 clones of M. bovis ATCC35721
(pYUB178: :M. bovis WAg200) were obtained and pooled. A
control electroporation of 400 ,ul organisms without added
plasmid DNA yielded no kanamycin resistant colonies.
Fifteen ~. bovis ATCC35721 (pY~1;3178: :M. bovi3 WAg200)
clones were selected before pooling and subcultured for
DNA preparation in 3-5 ml of the same media used for
culturing M. bovis ATCC35721. Genomic DNA of
recombinants, extracted by the method of van Soolingen et
al. (1991), was characterized by restriction fra3
digestion with PstI, electrophoresis, Southern blotting
and hybridization with a probe of pYU3178. This revealed
54
WO95/17~ 9~ PCT~S94/14912
the junction r _ t~ of the integrated cosmid and i8
referred to below as junction L I , t analygis . In all
cases the CL, t patterns were different.
E . Protocol to a35e99 Vi rl7l ~nre Qf tuber--l-l 08i5 coml~lex
The method for virulence testing in guinea pigs
was adapted from the procedures described in the Trudeau
My~r~h~ct~orial Culture Col l ec~ n catalogue, (Anon, 1972~ .
10 Albino, outbred guinea pigs were inoculated
subcutaneously in the f lank . Libraries and individual
strains of mycobacteria were inoculated into guinea pigs
which were kept in ~iltered-air, ventilated animal cages.
Animals were 3acrificed approximately 6 and 13 weeks
15 after in~culation and f.YAmi nl~fl for the presence of gross
lesions of tuberculosis. Samples from the in~ection
site, the prefemoral lymph nodes and spleen were cultured
for myct~hActpriA using previously described methods
(Collins and de ~isle 1984). ~ormalin-fixed tissues,
20 from the spleen, liver, kidney and lung were embedded in
paraffin, sectioned at 3-5 ~lm, and stained with either
hematoxylin and eosin (~E) or by the Ziehl-Neelsen
method .
i. First inocl~l~tion ex~eriments in quinea ~iqs
The level of virulence in guinea pigs of
M. bovis ATCC35721 was assessed by the sites in which
gross lesions were found (Table 3) . There were no such
lesions in the spleen. This indicated that ~. bovis
ATCC35721 was not sufficiently virulent to pass through
3 0 the lymph nodes draining the inj ection site and enter the
systemic circulation in sufficient numbers to cause gross
lesions to occur i~ the spleen.
2l79
Wo 95/17511 7 7~ PCI/US94/14912
TAB~E 3
Gross lesions in animals sacrificed 92 days after
infection with a 7 . 2 ml inoculum of M. bovis ATCC35721
containing 1. 9xlO colony forming units (CFU) .
5 Guinea pig Injection Prefemoral Spleen
site lymph nodes
A+ +
B+ +
10C +
In a subsequent experiment, the virulence of
the M. bovis ATCC35721 (pYUB178: :M. bovis WAg200~ library
was assessed at two time intervals and gross lesions were
15 identified as shown in Tables 4 and 5.
TABLE 4
Gross lesions in animals sacrificed ~0 days after
infection with a 0 . 2 ml inoculum of M. bovis
ATCC35721 (pYUB176: :M. bovis WAg200) library containin
approximately 10 CFU.
Guinea pigs Inj ection Pref emoral Spleen
Site lymph nodes
25A +/- +
B+ +
C+ + +
56
Wo 95/17511 2 ~ PCrlUS94ll4gl2
TABLE 5
Gross lesions in animals sacrificed 89 days after
infection with a o . 2 ml o inoculum of M. bovis
ATCC35721(pYUB176: :M. bovis WAg200~ library cnnt~;nin~
5 approximately 10 CFU.
Guinea pigs In]ection Prefemoral Spleen
site lymph nodes
A + + +
B + + +
C + + +
ii. Characterizatign of ~ ' ;n;lnt M. bovis
rom c~uinea ~iqs
Pre~emoral lymph node and spleen tissues of all
guinea pigs were cultured for the presence o M. bovis.
Apart from spleen tissue from guinea pig A in the 50 day
group, M. bovis organisms were isolated from all these
20 tissues. Over 160 individual clones repr~Rpn~i n~ all
lesion-cnnt~;ning prefemoral lymph nodes and spleens were
subcultured and their genomic D~A subjected to junction
fragment analysis. Approximately 80~ of all clones had
the same junction fragment pattern. Clones which gave
25 this pattern were found in all M. bovis cnnt:linin~
tissues. One o~ these ATCC35721 (pYUB178: :M. bovis
WAg200) clones cnnt~in;n~ the pre~, in~nt junction
fragment pattern designated as WAg300 was used for
further experiments below.
iii . Second inor~l ation experiment in quinea
~a
In this experiment the v' r~ nre of M. bovis
WAg300 ar~d ~ bovis ATCC35721 were compared concurrently.
Results are given in Tables 6 and 7.
57
Wo 95117511 2 1 7 ~ 7 7~ PCTIUS94114912
TABLE 6
Gross lesions in animals sacrificed 45 days after
infection with a Q . 2 ml inoculum of M. bovis ATCC35721
c-~n t ;I; n i n ~ 7 . 6xl 0 ~ CFU .
5 Guinea pigs Injection Prefemoral Spleen
site lymph nodes
A+ +
B+ +
10C +
TABLE 7
Gross lesions in animals sacrif iced 4s days af ter
infection with a 5.2 ml inoculum of M. bovis WAg300
15 c~nt~;nin~ 2.8xlO CFU.
Guinea pigs Injection Prefemoral Spleen
site lymph nodes
A+ + +
20B +
C+ + +
M. bovi6 strains isolated from these animals
25 were shown to be identical to M. bovis WAg300 by junction
f ragment analysis .
The difference between the two sets of guinea
pigs with respect to the presence or absence of spleen
lesions clearly indicated that M. bovis WAg300 was more
virulent than M. bovis ATCC35721.
F. Isolation of ~art of the inteqrated virulence
deterrr i n i n~T cosmid
Genomic DNA was prepared from M. bovis WAg300,
digested with the restriction enzyme Notl and ligated
under conditions favoring self ligation. The ligation
58
WO95/17SII 2~9~ PCT/US94/14912
mixture was electroporated into E. coli and kanamycin
resistant clones were ;Rnl~t~d. A plasmid isolated from
one of these clones was denoted pHUA2. This plasmid
nnntAinPd the pYU!3178 kanamycin resistance gene and
E. coli origin of replication from the integrated cosmid
in M. oovis WAg300 as well as approximately 6 kb of
co3mid insert DNA. The r~ tinnAh;r between pUHA2 and
the original cosmid, designated pUHA1, which was
integrated in M. oovis WAg300 and which was never
isolated in total is shown in Fig. 1.
G. Selection of ~n~mi ~c with ~ossibl~ vi rulence
determ; n; n~ f actors
A 2 kb Mlul fragment from the insert of p~HA2
was used as a colony hybridization probe of the E~. coli
pYU~3178: :M. bovis WAg200 library. Apprn~;r-t~l y one
colony in every 13~ library colonies gave a positive
hybri~;7~tinn signal. Cosmids were isolated from 48
hybridizing clones using standard plasmid preparation
methods and compared to each other and to pUH~2 on the
basis of restriction enzyme digestion ~ttf~rnC. Three
cosmids, designated pUHA3, pUHA4 and pUHA5, had most
similarity to pUHA2 and are shown in Fig. 2. Two other
cosmids with inserts which ov~rl ~rPed those of pUHA3 -
pUHA5 were also selected from the rr--;n;n~ 45 cosmids by
using pUHA2 as a probe of So~1th~rn blots of cosmid
restriction digests. These cosmids, designated pUHA6 and
pUHA7 are also shown in Fig. 2.
H. Pre~a~ation of ~utative virulence secuences for quinea
~i~ r~;nr~nl~l ~tion
Cosmids pUE~A3 -pUHA7 were ele- L ~ o.dted into
M. ~ovis ATCC35721 and clones of ~. bovis
ATCC35721 (pUHA3-pUHA7) were recovered using kanamycin
selection. These recombinant M. ~ovis clones, designated
WAg301-WAg311 were inoculated into guinea pigs to assess
59
W095117511 21 79772 PCrnlS94/14912
their virulence. The number of M. bovis clones
inoculated was greater than the number of cosmids because
in some cases, junction fragment analysis of individual
clones revealed three different patterns were obtained
5 f or some cosmids . In cases where more th~an one pattern
was obtained for DNA isolated from clones c nnt~;n;ng a
particular cosmid, subcultures of clones repr~Pnt;n~
each pattern were combined f or inoculation . The
association between cosmids and M. bovis recombinants is
10 shown in Table 1. Guinea pigs that had received M. bovis
recombinants ~-ont~;n;n~ cosmids p~EA3, p~A4, pUHA5, and
p~HA7 developed extensive lung or spleen lesions,
indicating that these cosmids had restored the virulence --
to the M. bovis ATCC35721 strain. These three cosmids
contain genomic inserts of approximately 40-43 kb and
have a common overlapping segment of approximately 10 kb.
Cosmid p~lA3 was partially digested by Sau3AI
and in separate experiments 2-4 kb and 10-15 kb fragments
were cloned into the cosmid shuttle vector pl~HA8. Vector
p~HA8 was produced from pYrJB17a by incorporating PacI
sites on either side of the ~3clI cloning site. These
libraries of p~HA3 were electroporated into ~. bovis
ATCC35721 to produced libraries of M. bovis
ATCC35721(p~HA8: :p~JHA3) . Approximately 300 rnlnn;~c from
the 2-4 kb library and 1000 colonies from the 10-15 kb
library were pooled separately, subcultured and
inoculated into guinea pigs.
Guinea pigs that had received ~. bovis
r--~ ;n~nt~ cnnt~;n;n~ either the 2-4 kb fragments or
the 10-15 kb ~L__ t~, developed extensive spleen
lesions indicating that the5e fragments had restored
virulence to the ~. bovis ATCC35721 strain. ~. bovis
organisms were isolated from the spleen le8ions and
subcultured for DNA extraction. DNA prepared from these
cultures was digested with PacI and electrophoresed on
wo 9s/l75~ 7 ~ 7 ~ ~ PCrlUS94/1491~ ~
agarose gels. No restriction ~Lcly, tR could be clearly
V; Rll~l; 7e~ by staining with ethidium bromide 80 the gels
were Southern blotted onto nylon and hybridized with a
DNA probe of the entire insert of pUH~2. This probe
5 revealed two hybridized bands for many of these isolates.
One of the bands was the same for all isolates and
corr~Rpnnrl~o~l to the pn9;t;nn on the blot of undigested
genomic DNA. The other band varied in size from one
isolate to another but in no case was smaller than
10 approximately 3 kb. One strain nnnts~;n;n~ an
approximately 3 kb fragment was designated WAg320 and
used for ~urther analysis. These results showed that a
DNA fragment of approximately 3 kb was sufficient to
restore virulence to M. ~ovis ATCC35721. This 3 kb
15 sequence has suf f icient overlap with the insert of pUElA2
for detectable hybridization to occur between them. This
alignment of the 3 kb sequence and pUHA2 is also
consistent with the virulence restoring abilities of
cosmids puHA4, pUHA5 and pUHA7 since most of the insert
2 o of pUHA2 is within the shared DNA segment of cosmids
pUE~A4, pUE~A5 , and pUHA7 .
I. Restriction ma~incl of ~UHA3 cosmid
A restriction map of cosmid plJHA3 ~Fig. 3 ) was
25 constructed for the enzymes MluI, Nhel and NotI using a
partial digestion technique. The cosmid insert rnnt:~;n~cl
no sites for the enzyme XoaI, whereas the p~}3178 vector
nnnt:~;n~l two sites as shown ~Fig. 3). In the technique
used, cosmid pUHA3 was partially digested with each of
3 0 the three enzymes separately and then the partial digests
were digested with XbaI. DNA f, _ t~ in each partial
digest were separated in duplicate by agarose
electrophoresis and transferred to nylon filters by
Southern blotting. One of the duplicates was hybridized
35 with a 32p labelled probe of the left hand vector arm of
61
~ WO95/17511 2179772 PCT/I~S94~14912
plJHA3 and the other duplicate was hybridized with a probe
of the right hand vector arm of pUHA3. Best estimates of
the molecular size differences between the labelled
fragments were obtained by comparison to labelled DNA
markers and these were also compared to L, ~ t sizes of
complete digests of pl~HA3 with the same enzyme.
J. Sequerlcinq of 3 kh sequence
WAg320 was digested with PacI and the 3 kb
fragment was ligated into the PacI site of the Se~F-nr; ng
vector pUHA9 using standard methods. The "Erase-a-base"
system (Promega) was used to make progressive,
unidirectional deletion mutants of two clones designated
pUHA11 and pUHA16 which c~nt~in~ the 3 kb fna~, in
opposite orientations. Appropriately sized deletion
mutants were cloned and chosen as instructed by the
manufacturer' 8 protocols . Polymerase chain reaction
sequencing was performed by using commercial kits ~Gibco-
BR~ and Intermed) in accordance with the manufacturer~ s
instructions. The 2745 bp fragment that restores
virulence to M. bovis ATCC35721 is shown in Figure 9.
Pigure 9A shows this sequence together with a 53 0 amino
acid translation of the largest ORF. The first codon of
this ORF at poaitions 835-837 i9 contiguous with the
likely ribosome binding site so ln;ti~tif,n may actuaily
occur at codon three at positions 841-843.
K. Coml~arison of the 3 kb Mvcobacterial DNA secluence
with GenBank sequences
The DNA sequence obtained from the 3 kb
fragment that restores virulence to M. }~ovis ATCC35721,
shown in Figure 9, was analyzed using the 7 . 3 . l-~NIX
- update (September 1993 ) of the program package supplied
by the University of Wisconsin Genetics Computer Group
35 (575 ^cienc~ Drive, Madison, Wisconsin 53711); this
Wo 95/17511 ~ 2 PCT71JS94/14912
package is abbreviated as "GCG". An earlier version of
the package is described in Devereux, J., et al., (1984),
N~al ~i rlc Res . 12: 387-395 .
The comparison was performed as follows. The
DNA sequences of the contigs were translated into amino
acids (using the program TR~NcT Z~T~) and compared to the
GenBank ~t~h~qe update ~2 O using the ~. uS~- TFASTA.
This comparison revealed that the sequence analyzed had
~ignificant homology with numerous sigma factors. Some
of the DNA s~ Pn~q of the sigma factors with which the
homology was particularly high were r~ht ~ i n~ri f rom the
GenBank database using the ~, UyL FETCH and their
coding sequences were translated into amino acids using
TR~NSLATE. These sigma factors were then compared to an
amino acid tr~nCl~tic~n (using TR~Ncr.~rrE) of the large ORF
on the largest contig using the ~, uy~ PILEUP. A
smaller downstream contig was also tr~nql ~t~l using
TR~NSLATE and compared in the same PILEUP, _ -~; qnn,
FETCH, PI~EUP, TFASTA and TRANCT ~Tr' are ~ U~ in the
2 0 GCG package .
The results of a PileUp comparison of hrdB
principal sigma factors from Streptomyces coelicolor
(GenBank ~c~q~si~n No. X52983) and Streptomyces griseus
(GenBank accession No. L08071) with the ami~o acid
translation of the ORF from the M. oovi3 virulence
restoring factor is shown in Figure 10-A. It can be seen
from the results that there is a high degree of
relatedness between all three sPq--~nrPa, particularly in
the region above 290.
Figure 11 presents the results of a GAP
comparison of Streptomyces griseus principal sigma factor
(Peptide translation of GenBank accession No. ~08071 from
nucleotide numbers 570 to 1907, which is the coding
sequence of the hrdB gene) with peptide translation of
the large ORF oi the apprn~ir~t~ly 3 kb DNA fragment from
63
21 ~97
WO95117511 72 pCr/US94/14912
M. bovis associated with virulence. Exact homology
between the sequences i8 indicated by vertical dashes.
While there were signif icant homologies of the
sequences encoded in the M. bovis Ll _ ~ with the sigma
5 f actor sequences indicated above, the overall homology
detected was less than about 65~ to 70~ with any specific: =
sequence. In addition, there was no exact match with any
of the Gen;3ank sequences
10 L. Identification of a Mutation Associated with
Avirulence
The 2.7 kb fragment from M. bovis WAg200 was
sequenced on both chains using an ordered deletion mutant
strategy and polymerase chain reaction sequencing with
15 33P. A probe of this fragment was used to select
hybridizing clones from replica plates of genomic
libraries of M. bovis ATCC35721, M. bovis WAg201 (another
virulent New Zealand strain), and M. tuberculosis Erdman.
The homologous DNA L~ were isolated and sequenced
20 and their large ORFs translated for the PILEUP
comparison .
The sequence of the 2.7 kb fragment ~nf~ofl;n~
the rpoV gene from M. bovis WAg200 and comparison of its
translation with those of other M. bovis and M.
25 tuberculosis rpoV genes and principal sigma factors from
two Streptomyces species is shown in Figure 12. Figure
12a presents the sequence of M. bovis WAg200 showing the
large ORF which begins with GTG at position 835-837
Since the potential ribosome binding sites (underlined)
30 are so close or overlap this codon, the likely initiation
site is the third codon of the ORF, as indicated. The
three mutations in M. bovis ATCC35721 and their e~fect on
the translation of rpoV are shown respectively above and
below the equivalent sequences from M. bovis WAg200 Two
35 of the three mutations are also found in one or more of
64
Wo 95117511 PCrnJS94/14912
2~,~9~2 ' '
the other M. tllberculosis complex strains analyzed
(fitrain numbers in brackets) .
Figure 12b presents a comparison of putative
principal sigma ~actors of ~our M. tuberculosis complex
5 strains and two Streptomyces sp. Upper case letters
denote amino acids that agree with t~e consensus sequence
of the M. tuberculosis complex. An arrow denotes the
position o~ the amino acid in the M. bovis ATCC35721
sequence that differs from that of all three of the other
o M. tuberculosis complex strains. The3e results in~icate
that it is this difference that causes M. bovis ATCC35721
to become avirulent. This position is highly conserved
among principal sigwa factors and their homologues and
the region in which it occurs has the characteristics of
15 a helix-turn-helix motif and is believed to be involved
in -35 sequence recognition. (Lonetto, M. et al. (1992),
J. Bact. 174:3843-3849) . Mutation of an arginine to a
histidine in this region has previously been shown to
cause an alteration in promoter recognition in
20 E~schicherichia coli (Gardella, T., et al. ~19a9), J. Mol.
Biol. 206:579-590) . But, tA~irn at the equivalent
position in the M. bovis ATCC 35721 sequence has not been
reported .
Exaw~le 2
poI,yNurT~r~TTnr~q ENCODTNG vrRrTr~r~NcE FACTORS ISOLATE~ B~ A
MOUSE COMPT r~ENT~TIoN }~qq~v
A method f or identifying virulence determinants
by genetic complementation was discovered that reguires:
(i) two strains that are genetically similar; ~ii) a
phenotype a~sociated with virulence; and (iii) gene
transfer systems. An existing pair of M. tuberculoçis
strain3, H37Rv (virulent) and H37Ra (avirulent),
distinguishable by their ability to cause disease in
3s animal models were used. H37Ra and H37Rv were derived
6~
WO 9S/17511 ~1 7~ 7 72 PCT/US94/14912
from the same clinical isolate in 1934 and pulsed field
gel analyses of D~A fragments generated by digestion with
inf requently cutting enzymes revealed that their
macroscopic genome orr,anization was similar. The
5 well-characterized difference in growth rates in mouse
lungs and spleens of H37Ra and H37Rv correlated with
their pathogenicity. The ability of H37Ra/H37Rv
rPro~inAntg to grow faster than H37Ra in the mouse was
def ined as a potential virulence phenotype .
A genomic library of M. tuberculosis H37Rv was
constructed in an integrating cosmid vector, pYL~;3178, and
electroporated into H37Ra. Mice were lnfected with pools
of FB7Ra recombinants rr,ntAin;ng H37Rv DNA to allow the
selection of growing clones in mouse spleen and lung.
15 The integrating shuttle cosmid libraries, based on the
mycobacteriophage L5 ; ntegrat; on system, were ideal or
in vivo rr.n~rl ~ ' ation because: ~i) only approximately
225 clones were rer~uired to represent the H37Rv genome,
(ii~ toxic effects associated with the expression of
20 genes from multicopy plasmids were avoided,
(iii) kanamycin selection pressure was not necessary to
maintain the cosmid, and (iv) clusters of contiguous
genes can be delivered and expressed.
The growth rates of selected recombinants were
25 measured in mouse spleen and lung, and a method was
developed to retrieve the H37Rv insert DNA from the
chromosome of a recombinant. This method allowed for the
~ nt;f;c~at;on and characterization of a 25 kb DNA
fragment of ~. tuoerculosis which conferred an in vivo
30 growth advantage to the growth-defective H37Ra.
A. Bacterial strain3 and r~rowth crn~; tions
M. tuberculosis strains H37Ra and H37Rv were
provided by Wilbur Jones of the Centers for Disease
35 Control, Atlanta, and were grown in enriched 7H9 broth
WO95/17511 ~ 9P~2- PCr~S94/14912
[Middlebrook 7~9 medium enriched with albumin-dextrose
complex (ADC) or oleic acid-albumin-dextrose complex
(OADC) (Difco ~aboratories, Detroit, Mich.) and a 0.05
polyoxyethylene sorbitan monooleate (Tween-80~1') ], under
siosafety Level 3 (s5L3) f~nn~:~i t. All cultures were
grown at 37C. E. coli strains X2764 (13), HB101 (4) and
DE~5~ (RPthP~ Research Laboratories Life Te~hnnl ~sies
Inc., Gaithersburg, MD) were grown in L broth. Strain
X2764 was grown at 30OC. See Table 8 for a list of
strains and plasmids.
B . Construction of shuttle co, ~-; d Anrl T~7Rv 1~ hr;~ry
The pYUB178 integrating shuttle cosmid
(Figure lA), was constructed by ligating the 975 bp
co8-''nntA;n;n~ BglII/BclI fragment of lambda DNA to the
BclI-digested, calf-intestine alkaline phosphatase (CIP)-
treated (Boehringer Mannheim BiochemicalEi, Tnrl;AnArolis,
IN) pMV305.F (18, 27) under conditions which favored the
formation of linear cnnr;qt: rS, i.e. greater than 50
ng/~l final DNA nnnl-Pntration.
Genomic DNA of H37Rv was prepared by -hAn; rAl
disruption of bacterial cell~ and subse~uent
phenol-cloroform extractions as previously described
(12) . ~37Rv genomic DNA wa3 partially digested with a
range of rnnt-Pntrations of Sau3AI to generate 30-50 kb-
sized fL~I a . F~ _ nt~ of 30-50 kb were; ao1 ~t~l as
previously described (14). The 30-50 kb Sau3AI fragments
of chr, ~ 1 DNA were then ligated to CIP-treated,
BclI-digested pYUB178 DNA; the final DNA rnnnpntration
was 50-100 ng/lll and the DNA molar ratio of insert to
vector was 1.
C . T.; hrAry T:~ackaqinc into lambda ~haae heads and tails
Four ~1 of a- ten ~1 ligation mixture was in
vitro-packaged with the GigaPack II Packaging E:xtract
67
WO95/17SI1 21 79 ~2 pCr/US94/14912
(Stratagene, La Jolla, CA) according to the
manufacturer's procedure. The in vitro-packaged lysate
was tran~ red, using previously described methods (14),
into the in vivo packaging strain oi- E, coli X2764 (13).
D. ~n vivo-~acka~in~
The 103-104 kanamycin-registant rP~ in~nt
clones were pooled and inoculated into L broth ~ nn~; n;n~
25 ~g/ml kanamycin. One aliquot was grown to prepare
plasmid DNA by an ~lk~l;nf~ lysis method. The other
aliquot was grown by in vivo-packaging which was
accomplished by previously described procedures (13).
The titer of the lysate prepared from X2764 transductants
cnnt~in;n~ the pYU;3178: :~I37Rv library was approximately
1 x 109 cfu/ml. The lysate was stored at 4C after
filtering through a 0.45 ~m pore sterile ~ilter.
E. Con3truction of H37Ra (~Y~3178: :H37Rv) recombin;~nt
An eight day old H37Ra culture was
electroporated with the pYU;3178: :H37Rv library DNA in
plasmid form, and separately, with pYU~3178 DNA.
Approximately 450 transformants arose from five
independent electroporations of cells with apprnYi~-t~ly
1 llg library DNA each. Two pools of H37Ra
(pYU~3178: :H37Rv) rer ' in~nts, pool 1 and pool 2, were
made by collecting and inoculating approximately 225
colonies into 50 ml of enriched 7H9 broth cnnt~inin~ 10
~Lg/ml kanamycin, and allowing growth for approY;~tF~ly
two weeks. Aliquots of pools were distributed and frozen
in cryovials for later use in animal experiments.
Another pool of H37Ra(pYU~3178: :H37Rv)
recombinants, pool 3, consisted of approximately 260
clones and was used to determine whether the pools were
representative. R.-cr~ in~nts of pool 3 were collected
directly from plates of enriched Middlebrook 7H10 agar
68
Wo 95/17511 2~ ~ grt ~ ~ PCrlUS94/14912
c~"t~inin~ 25 /lg/ml kanamycin after growth following
electroFor~t;on; an ali~ ot was inoculated into enriched
7Hg broth without kanamycin and allowed to grow standing
at 37C for approximately two weeks. Total D~A was
5 ; f:Ol ~t.--l from pool 3 before and after growth in broth.
DNA was subjected to Southern analysis using the l.l. kb
DraI/SspI DNA fragment oi pYU13l78 as a probe.
F. Mouse infection
In experiments J2, J2P, J5 and J5P that used
the mouse to select individual recombinant clones rom
E1ools 1 and 2, and in subsequent growth mea~u,~ t
experiments, J33 and J~6, groups of C57BI./6 mice aged 6-8
weeks were intravenously inoculated with 0 . 2 ml of each
culture tested . Five mice were i nnclll at~d with each
rP~ ' ;n~nt group or control group per timepoint.
Tn~ ti~n of mice with spleen-p~s~e~ bacteria was
~c~mrl; ~l~A by first homogenizing spleens after fourteen
days infection in 5 ml sterile saline. One ml of the 5
ml spleen homogenate from each of the five mice per group
was pooled and f iltered through sterile gauze to exclude
tissue clumps . The f iltrate was used to directly
inoculate another set of mice in experiments J2P and J5P.
See Table 9 for details or mouse experiments.
Individual colonies that grew from plated lung
homogenates in experiments J2P and J5P were picked and
grown in enriched 7E~9 broth f or subsequent mouse
experiments and DNA analyses.
C. R~triev~l of ~UB178: :H37Rv Cogmidg from r ~ 0 =R
of in vivo-selected re~ ~in~nts
Chromosomal DNA was isolated from individual
H37Ra (pYUB178: :H37Rv) recombinant clones using chemical
disruption of bacterial cells as previously described
(28~. DNA was partially digested with Sau3AI; r,C~. t~:
of 30-5D kb were size-fractionated and eluted from
69
~ 7~
WO 95/17511 7 72 PCrlUS94/14912
agarose gels as described above. The 30-50 kb fragments
were ligated to the 975 bp BglII/BclI fragment r~nt;~;nln~
cos of coliphage lambda DNA. The ligation conditions
were such that the final DNA cnnr.ontration was 50 to 100
ng~l, and the molar ratio of ~ q~ 1 DNA fragments
to co~ DNA rL~ ~ was 1.
The ligation mixture was packaged into lambda
phage heads and tails using the Stratagene GigaPack kit,
and trAn~ s~rl into E. coli strain HB101. Individual
kanamycin-resistant transductant colonies were picked and
cosmid DNA was isolated. Cosmid DNA was then analyzed by
restriction digestion and Southern hybr;tl;7at;nn.
H. Restriction and Southern ;ln~lv8e8 of retrieved cosmids
Digested cosmid DNA was subjected to agarose
gel electrophoresis in 0 . 8~ agarose in TAE buffer. DNA
was Southern blotted from gels onto nylon membranes by
capillary diffusion, W-crosslinked and hybridized with
probes derived from pYUB178. Probes consisted of either
the 1.1 kb LlraI/SspI fragment of pYU~3178, or the 436 bp
AseI/sclI LL _ t of pYUB178 that contained lambda DNA
adjacent to CO6, or the 756 bp AseI/ElclI fragment of
pYUB178 that ~-nnt~;n~1 part of aph. Probes were labeled
with {~-32P}dCTP using random hexamer priming with the
Pharmacia nl;gol~h~l ;ns kit ~Pharmacia LKB Biotechnology
AB , Uppsala , Sweden), or with horseradish peroxidase
according to the protocol of the Fnh~nC~d
Chemill~m;n~-~c~nre ECL Gene Det~ct;nn System (Amersham
Tntf~rn:ltional, Amersham~ UR).
I. Scr,-~n;n~r the ~YUB178: :H37Rv l;hr;~rv in E. coli
The pYUB178: :H37Rv library DNA lysate, 109
cfu/ml, was serially diluted to a cnnr~ntration o 104
cfu/ml in SM buffer [50 mM Tris-Cl (pH 7.5), 100 mM NaCl,
8 mM MgS04 7H20], and tr~n~ d into E. coli strain
WO95/17511 , ~ 9~ Pcrluss4ll49l2
HBlOl. Aliguots of infected cella were plated onto B
agar ~nn~;n;n~ 25 ~g/ml kanamycin such that each plate
would contain approximately l50 colonies. After
overnight in~llh~tinn at 37C, I-nlnn;.~q ~rom each plate
5 were lifted onto Biotrans nylon filters ~ICN Biomedicals,
Inc ., Irvine , CA) . The f ilters were treated with
denaturing buffer and neutralization buffer and W-
crosslinked. A probe was made from a cosmid, pYUB352,
derived from the mc2806 recombinant clone. The cosmid
10 pYUB352 was linearized by digestion with AseI and labeled
with [cy_32p] dCTP . Filters were hybridized overnight
according to the manufacturer' 8 protocol ~ICN
, Inc. ) .
Thirty hybridizing clone8 were picked and
15 streaked onto plates, and subjected to secondary
screening with the pYUB352 probe. Ten strongly
hybridizing clones were picked and analyzed by Southern
hybridization with pYUB352 as a probe. Four cosmids, two
that shared X37Rv restriction fragments with pY~3B352, and
20 two that did not share H37Rv restriction fragments with
pYUB352, were electroporated individually into X37Ra.
J. In vlvo qrowth of ~YUB352-overl ~n~inq and
-~onoverla~2inq recombinant3
Single H37Ra transformant colonies from each o~
the f our electroporations were grown in enriched 7}~9
broth cnnt~;n;n~ kanamycin to prepare sufficient culture
for mouse experiments. The in vivo growth rates of H37Ra
~nnt::l;n;n~ pyuB352-overlapping and -nonoverlapping clones
were measured in the experiment designated J36 ~see Table
9) .
71
W095117511 217977,~ PCr/US94114912
R. Res111 ts
i . Construc~ion of shuttle co.c~; tl ~nt~ H37Rv .
librarY
The in~e~t;n~ cosmid p~Bl78 t~t~n~;nR an E.
coli ori derived from plJCl9, the L5 attP site, the L5
integrase gene, a kanamycin resistance gene, aph, derived
from Tn903, the lambda cos se~auence and a unique cloning
site, BclI (see Figure 4A). The L5 mycobacteriophage
lO att~t' ~ site attP, and integra6e gere, int, mediate
site-specific integration into the mycobacterial
C11LI 3: (18) . The H37Rv library was constructed by
ligating 40 kb size-selected chromosomal DNA fragments,
ge~erated by partial digestion with Sau3AI, to ~lki~l;nt~
15 phosphatase-treated pYUBl78, linearized by digestion with
BclI. The ligation mix was packaged into lambda phage
heads and tails, and transduced into E. coli. The
approximately 4000 kanamycin-resistant transductant
colonies were theoretically enough to represent the B 7Rv
20 genome forty times. Twelve individual cosmids of the
H37Rv library were isolated from randomly picked E. coli
transductant colonies and examined by restriction
analyses. No two cosmids were alike, and each cosmid had
an insert size of 35-40 kb (data not shown). The B7Rv
library DNA was isolated as plasmid from the complete ~-
pool of E. coli transductants and elet_LL~uLated into
H37Ra. To identify the H37Rv insert within the
chromosome of a H37Ra(pYUBl78: :H37Rv) recombinant, a
method to detect the B7Rv DNA fLo~ ' R; ~ tt~ly
adjacent to pY~Bl78 sequences was devised. The method of
analysis depicted in Figure 4B allows the ; t~tsn~ i f; t~ation
of PstI restriction fragments of the H37Rv DNA at the
junctions of pY~3178 8t~1t'nt~t~R on either side of the BclI
cloning site (see Figure 4B). The pY~lBl78-H37Rv
junctional fragments of individual H37Ra (p~UBl78: :H37Rv)
72
Wo 95/17511 ~ ` PCT/US94/14912
recombinants are visible as bands in the Sollthl~rn
analysis in Figure 4C/ lanes 1-3.
To determine if a ~ LI~RF-llt~tive panel of
H37Ra (pYUB178: :EI37Rv) recombinants was generated,
S apprn~ t~ly 260 transformant colonies, pool 3, were
collected after growth on kanamycin-rnnt~in;n~ 7X10 agar;
an aliquot of pool 3 was transferred to enriched 7H9
medium and allowed to grow for apprnY;m-tf~ly two weeks.
C~ 1 DNA was isolated irom pool 3 both before and
10 after growth in broth. These DNAs were subjected to P3tI
digestion and agarose gel electrophoresis, followed by
transfer to a nylon membrane and hybridization to a
pY~3178 probe (Figure 4C). In figure 4C, the smears in
lanes 4 and 5 reveal that the pool of
H37Ra ~pYU!3178: :H37Rv) recombinants co~sisted of members
having different X37Rv DNA inserts, both before and after
growth in broth, suggesting that the pools were
repr~rnt~t;ve and stable in the absence of kanamycin
selection pressure.
ii. Enrichment and selection of ~utativelv
V; r~ nt rernmhin~nt~ from ~0018
Mice were intravenously inf ected with either
H37Ra(pY~3178: :~37Rv) recombinant pool 1 or 2 Two weeks
post-infection, mouse spleens were individually
hnn~ n; 7e~ pooled, and used to infect a second group of
mice. Individual recombinant rolnn;~ç~ that grew from the
plated lung h~ , -t~ prepared from the second group of
mice were picked. To characterize the integrated cosmid
in each recombinant, ~:hL I ~ 1 DNAs were isolated f rom
these individual rP~~ ' ;n~ntS and subjected to So~lth~rn
analysis with a pY[~3178 probe. The junctional fragment
analyses of selected individual L~ -' ;n -nt~ from the in
vivo-passed pool 2 in experiment J5P (see Table 9) are
shown in Figure 4C, lanes 1, 2 and 3. Lane 1 shows the
clone designated mc2807, lane 2 shows the clone
73
W09S117511 21 79 77~ PCIIIJS94114912
designated mc2806, and lane 3 shows a clone that has
junctional fragments identical to those of mc2806.
Because clones having junctional fldl - -q iri~ntir~l to
those of mc2806 were isoIated from many animals during
5 two different e~cperiments, J2P and J5P, (data not shown),
mc2806 was further characterized.
iii. In vivo qrQwth rate cn~risons
Growth rate comparisons of clones mc2806,
mc2816 (H37Ra f~ont;~;nin~ pYUPl78, see Table 9) and H37Rv
were made (see Figure 5). Clone mc2806 grew in the
spleen at a rate that was slightly lower than the growth
rate of H37Rv during the first two weeks of infection.
Clone mc28l6 barely grew. After the initial growth
phase, mc2806 was cleared from the spleen at a lower rate
than the rate of clearance of mc28l6. H37Rv persisted at
its day 28 level, at least through the experimental
endpoint, day 84. Clone mc2806 did not grow faster than
mc28l6 during the first two weeks in mouse lung (Figure
5~3). Thereore the faster i~ vivo growth rate of mc2806
compared to mc28l6 was evident only in mouse spleen. The
growth rates of mc2806, mc28l6, and H37Rv in enriched 7H9
broth were Yirtually ;~ont;~l (data not shown).
iv. Identification of a H37Rv DNA inqert that
confers a aster in vivo qrowth rate to H37Ra
To prove that the H37Rv DNA insert present ir
an in vivo-selected recombinant was responsible for its
in vivo growth phenotype, it had to be retrieved rom the -
c1.~, - ~. A disadvantage of the stably integrating
pYU~3178: :~37Rv cosmid library i9 the difficulty of cosmid
retrieval from the ~ LI ~_ ~ of a H37Ra (pYU~3178: :H37Rv)
re~ ; n5~nt; the excision functions of L5 are not yet
understood. ~lence, a method was devised to clone the
H37Rv DNA insert as a cosmid (see Figure 6A) . The lambda
35 in vitro-packaged 1 ;~tinn mix that cnnt;l;nF.~ random
Wo 95117511 PCr~7S94/14912
~,~7~
pieces of the mc2806 I-11L~ ~ was trAnc~7~ 7 into E.
coli for the purpose Of ~qPlr~t;n~ H37Rv DNA-cnnt-7;nin~
cosmids. Only those cosmids rnnt 7inin~ the E. coli and
aph replicated under kanamycin selection pressure (cf
5 Figure 6A). Tke Southern analyses of 16 of the 33
retrieved cosmids of mc2806 from E. coli trAnqr7llctAn~tq is
shown in Figure 6B. The cosmids were digested with both
EcoRI and AseI and analyzed by gel electrophoresis. The
434 bp probe, generated by digestion o~ pYI7Bl78 with AseI
lo and 7clI, hybridized to the H37Rv/p~7B178 junction that
included lambda DNA adj acent to cos . By comparing the
sizes of the junctional L. _ ~c of the retrieved
cosmids with the sizes of the junctional ~L_._ ' q. of
mc2806 in lane 1, one can determine whether the entire
H37Rv insert DNA has been retrieved. Cnly one of the 16
cosmids did not contain the full-sized H37Rv fragment
adjacent to the pYI.7B178 junction ~Figure 6B, lane 14).
The retrieval procedure was very e~icient; 32 of the 33
mc2806-retrieved cosmids ~nntA;npd the entire d37Rv
insert (data not shown) . The cosmid clone designated
p~3352 i~l la~e 15 was used for further study.
v. Identification of ~YIr!3352-overla7~7~inq
, ncmi ,7c from t7~e 7~ 3178: :H37Rv DNA librarY
To prove that the EI37Rv insert DNA was
responsible for the spleen growth phenotype, it had to be
reintroduced into X37Ra and tested. Reintroduction of
the H37Rv insert DNA from the mc2806 recombinant into
H37Ra required a replicating vector. Retrieved cosmids
did not have the ability to replicate in mycobacteria
3 because they lost the int gene when they were removed
from the CilL. -~ of the r~l ' in 7nts. Therefore,
pYI7B352 DNA was used as a probe to screen the
pYr B178: :H37RV library in E. coli for the H37Rv DNA
insert associated with mc2806. Colonies of E. coli
(pYI7B178: :~37Rv) library trAnc~7l7rt~ntc were transferred
~ Wo95~17511 21 79 77~ PC'r/US94/14912
to nylon filters, lysed, and probed with pYU~3352 DNA.
Cosmids that shared H37Rv DNA with pYU~3352, designated
pYtr~3353 and pYU3354, and unrelated cosmids, designated
pY~3355 and pYC13356, were separately transformed into
5 H3 7Ra .
vi The E~37Rv DNA Qf mC2806 c~lnfers ~ vivo
qrowth advantar~e tQ E~37~
The growth rates o_ H37Ra recombinant clones
rrntA;n;nS p~;3352-overlapping and -nonoverlapping
10 cosmids were te3ted in mice (experiment J36, see Table
9) . The H37Ra rerr~;n~ntC c~nt~;n;nrJ the pYU~3352-
overlapping cosmids grew as well as mc2aO6, and the H37Ra
recombinants ront~ininrJ pYt~;3352-nonoverlapping cosmids
grew poorly or did not grow at all (Figure 7~. These ~ -
data indicate that the H37Rv DNA that is shared by
pYU;3352, pYU~3353, and pYUF3354 expresses a gene or gene(s)
associated with growth in the spleen.
vii . M~nni nq the ivq ~eqion gf H37Rv
The pYtl;3352, pYU3353, and pYU335~ cosmids were
mapped by restriction digest and analyzed by Southern
hybridization (see Figure 8). The schematic of Figure 8C
shows the physical map of the H37Rv DNA insert of each
clone. A DNA region of apprrYir-t~oly 25 kb is shared
25 between the clones. This region was designated ivg or in
vivo growth advantage.
76
WO 95117511 ~ PCr~S94/14912 *
TABLE 8
Bacterial
strain or clone Description Source
5 E. coli
HBlOl F-aral4 leuB6 proA2 (3)
lacYl glnV44 gal~21-
recA13 rp3L20 xyl-5
mtl-1 thi-1 hsdS20
2 7 6 4 HBl û l lysogeni zed ( 8 )
X with A c1857 b2
red,~3 S7
DH5~ F-endAl hsdR17 BRL, Inc.
supE44 thi-1 l-recAl
gyrA9 6 relAl
(argF-laczya) Ul69
~P80dlacZ ~Ml5
M . tubercul osi s
mc2806 H37Ra cont~inin~ This study
pYUBl78: :H37Rv ivg
mc2822 H37Ra t~nnt~;ning This study
pYUB3 5 3
mc2823 H37Ra cnnt~;ning This study
pyUB3 54
mc2824 H37Ra cnnt:~inin~ This study
pYUB3 5 5
mc2825 H37Ra cnnt~;n;n~ This study
2 5 pYUB3 5 6
Shuttle Plasmid
YUBl78 Integrating shuttle This study
cosmid vector
pYUB352 H37Rv ivg-cnnt~;nin~ This study
3 o co2mid derived ~rom
mc 806
pYUB353 _ pYUBl78: :H37Rv ~vg This study
pYUB354 pYUBl78: :H37Rv ivg This study
pYUB355 pYUBl78: :H37Rv This study
35 pYUB356 pYUBl78: :H37Rv T~is study
77
WO9S/17511 17~77~ rCT/US94114gl2
TA~3~E 9
Experiment Pools and Inocula Timepoints
Cl one 5 ( c f u/mous e ) ( day )
Tested
5 J2 Pool 1 2 x 105
- Po2ol 2 6 x 105 1, 14, 28
mc 816 1 x 106
J5 Pool 1 1 x 1 0 5
2 6 x 105 1, 14, 28
mc 816 1 x 1o6
H37Rv 6 x 104
*J2P Pool 1 5 x 102
Poo21 2 7 x 102 1, 14
mc 816 5 x 102
*J5P Pool 1 9 x 102
Po2ol 2 7 x 102 1, 14
mc 816 6 x 103
J33 mc2806, 1-2 x 404
mc2816, 4 x 10 1, 14, 28,
H37Rv 5 x 104 84
J36 mc2806, 1 x 104
mc2822, 1-2 x 104
mc2823, 1-3 x 104
mc2824 5 x 104 2, 14, 28
mc2825, 6 x 104 87
mc2816, 8 x 104
H37Rv 4 x 104
*For J2P and J5P, inocula were e3timated ~rom cfu
25 retained in the spleen on day 1; spleen r~t~nt;on is
usually 10~ of the ;nr~cll12(t-;n~ dose.
78
