CA2179772A1 - Mycobacteria virulence factors and a method for their identification - Google Patents

Abstract

The present invention provides polynucleotides associated with virulence in mycobacteria, and particularly a fragment of DNA isolated from M. bovis that contains a region encoding a putative sigma factor. Also provided are methods for a DNA sequence or sequences associated with virulence determinants in mycobacteria, and particularly in M. tuberculosis and M. bovis. The invention also provides corresponding polynucleotides associated with avirulence in mycobacteria. In addition, the invention provides a method for producing strains with altered virulence or other properties which can themselves be used to identify and manipulate individual genes.

Description

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.

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 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 ~ 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 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 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 `~ 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.

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.

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.

-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 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 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 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 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 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 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 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 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 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 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 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 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 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~

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 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 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 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 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 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.

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+ + +

Wo 95/17511 2 ~ PCrlUS94ll4gl2 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.

Wo 95117511 2 1 7 ~ 7 7~ PCTIUS94114912 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 +

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 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 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 ~ 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 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 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 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 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 ~ 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) .

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) 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 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.

WO 95117511 ~ PCr~S94/14912 *

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 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.

Claims (21)

WE CLAIM:

1. A method for identifying a DNA sequence 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 comprising the steps of:
a) preparing a genomic DNA library of the pathogenic organism;
b) constructing an integrating shuttle vector containing genomic inserts prepared in step a);
c) transforming via homologous recombination a 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 g) identifying the DNA insert which confers virulence.

2. A method according to claim 1 wherein the individual inoculated is a mouse.

3. A method according to claim 1 wherein the individual inoculated is a guinea pig.

4. An isolated polynucleotide comprised of a segment of less than 3kb that is essentially homologous to a mycobacterial DNA sequence associated with virulence in mycobacteria, wherein the mycobacterial DNA sequence encodes a sigma factor.

5. An isolated 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.

6. An isolated polynucleotide according to claim 5, wherein the polypeptide is essentially homologous to the polypeptide encoded in Figure 9.

7. An isolated polynucleotide comprised of at least 15 sequential nucleotides homologous to a sequence of polynucleotides in Figure 9.

8. A recombinant polynucleotide comprised of a sequence of at least 15 sequential nucleotides homologous to a sequence of polynucleotides in Figure 9.

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.

10. An expression vector comprised of the recombinant polynucleotide of claim 9.

11. An isolated polynucleotide comprised of a linear segment of at least 15 nucleotides that is substantially homologous to mycobacterial DNA in a plasmid selected from the group consisting of pUHA1, pUHA2, pUHA3, pUHA4, pUHA5, pUHA6, pUHA7, pUHA11, pUHA16, pYUB352, pYUB353, and pYUB354.

12. A host cell comprised of a polynucleotide selected from the group consisting of the polynucleotide of claim 1, claim 2, claim 3, claim 4, claim 5, claim 6, claim 7, claim 8, and claim 9.

13. A host cell comprised of a polynucleotide according to claim 11.

14. A host cell comprised of the expression vector of claim 10.

15. A diagnostic kit comprised of a polynucleotide and a buffer packaged in suitable vials, wherein the polynucleotide is selected from the polynucleotides according to claims 3, 4, 5, 6, 7, 8, and 9.

16. An isolated polypeptide substantially homologous to a polypeptide associated with virulence in mycobacteria or a fragment thereof, wherein the mycobacterial polypeptide is a sigma factor.

17. The isolated polypeptide of claim 16, wherein the mycobacterial polypeptide is encoded in a DNA
sequence shown in Figure 9.

18. An isolated polynucleotide comprised of a segment of less than 3kb that is essentially homologous to a mycobacterial DNA sequence associated with avirulence in mycobacteria, wherein the mycobacterial DNA
sequence encodes a sigma factor.

19. A method for producing an altered property in a wild-type bacterial strain other than M. bovis comprising mutagenizing a principal sigma factor in the bacteria, wherein the mutagenizing results in converting an arginine to a histidine in the principal sigma factor, and wherein the conversion occurs at a similar position to that present in M. bovis ATCC 35721.

20. The method of claim 19 wherein the mutagenizing results in altered virulence properties of the resulting bacterial strain.

21. A method of using a bacterial strain prepared by the method described in claim 20, the method comprising preparing a vaccine by mixing a pharmacologically effective dose of the strain with a pharmaceutically acceptable suitable excipient.