EP0736098A1 - Mycobacteria virulence factors and a method for their identification - Google Patents
Mycobacteria virulence factors and a method for their identificationInfo
- Publication number
- EP0736098A1 EP0736098A1 EP95906122A EP95906122A EP0736098A1 EP 0736098 A1 EP0736098 A1 EP 0736098A1 EP 95906122 A EP95906122 A EP 95906122A EP 95906122 A EP95906122 A EP 95906122A EP 0736098 A1 EP0736098 A1 EP 0736098A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- virulence
- dna
- polypeptide
- bovis
- mycobacteria
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
- A61P31/06—Antibacterial agents for tuberculosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Definitions
- This invention relates 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 sequences.
- the mycobacteria are rod-shaped, acid-fast, aerobic bacilli that do not form spores.
- mycobacteria are pathogenic to humans and/or animals, and determining factors associated with their virulence are of prime importance.
- 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 pathogenesis .
- the disease is caused by infection with Mycobacterium tuberculosis; tubercle bacilli are inhaled and then ingested by alveolar macrophages. As is the case with most pathogens, infection with M. tuberculosis does not always result in disease.
- CMI cell-mediated immunity
- M. bovis causes tuberculosis in a wide range of animals including humans in which it causes a disease that is clinically indistinguishable from that caused by M. tuberculosis .
- Human tuberculosis is a major cause of mortality throughout the world, particularly in less developed countries. It accounts for approximately eight million new cases of clinical disease and three million deaths each year. Bovine tuberculosis, as well as causing a small percentage of these human cases, is a major cause of animal suffering and large economic costs in the animal industries.
- Antibiotic treatment of tuberculosis is very expensive and requires prolonged administration of a combination of several antituberculosis drugs. Treatment 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 becoming more 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, isoniazid and rifampicin. Treatment of these patients presents extreme difficulty and in practice is often unsuccessful.
- Bacterial RNA polymerases are composed of a core enzyme with the subunit composition anc - one °-** 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 classes of promoters.
- the principal sigma factor plays a central role in bacterial by promoting essential "housekeeping" genes. Genes for alternative sigma factors are present in all bacteria and have been shown to promote specific virulence genes in some pathogens (Fang, 1992; Deretic 1994) . However, loss of a virulence phenotype due to mutation in a principal sigma factor has not been reported. Streptomyces 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.
- Mycobacterial virulence Virulent strains of Mycobacterium tuberculosis have faster in vivo doubling times and are better equipped to resist growth inhibiting functions of the macrophages in the presence and absence of specific immunity
- the present invention provides isolated and recombinant polynucleotide sequences associated with virulence determinants in members of the genus mycobacteria, particularly those of the tuberculosis complex, and more particularly in M. tuberculosis and M. Jbovis. Based upon homology to sigma factors from other microorganisms, one of the mycobacterial sequences associated with virulence encodes a putative sigma-like factor.
- the DNA sequences encoding factors associated with virulence were found by the use of in vivo complementation assays, more particularly by complementation in a guinea pig model and in a mouse model .
- the in vivo genetic complementation systems utilized integrating shuttle cosmid libraries to identify potential virulence genes.
- the invention also provides techniques to identify 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.
- embodiments of the invention include the following.
- 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.
- This method may be performed with individuals that are mice or guinea pigs.
- the polypeptide may be essentially homologous to the polypeptide encoded in Figure 9.
- An isolated polynucleotide comprised of at least 15 sequential nucleotides homologous to a sequence 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 substantially homologous to mycobacterial DNA in a plasmid selected from the group consisting of pUHAl, pUHA2, pUHA3, pUHA4, pUHA5, pUHA6, pUHA7, pUHA8, pUHA9, pUHAll, pYUB352, pYUB353, and pYUB354.
- 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.
- mycobacterial polypeptide substantially homologous to a polypeptide associated with virulence in mycobacteria or a fragment thereof, wherein the mycobacterial polypeptide is a sigma factor.
- the mycobacterial 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 in mycobacteria, wherein the mycobacterial DNA sequence encodes a sigma factor.
- 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.
- This method includes altering the virulence properties of the bacterial strain.
- Figure 1 is a schematic illustrating the strategy for recovering part of cosmid pUHAl from M. bovis WAg300 which is a member of the M. bovis ATCC35721 (pYUB178 : : 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 pUHA2-pU__A7 in linear form for comparison purposes beginning with the NotI site at position 2024 of pYUB178.
- Cosmids pUHA3-PUHA7 were isolated by colony hybridization using a probe of the 2 kb Aflul fragment of PUHA2 : M, Mlul site; N, NotI site;
- Figure 3 is a restriction map of cosmid PUHA3 in linear form starting with the NotI site at position 2024 of pYUB178:h, Nhel; M, Mlul; ⁇ , NotI , X, Xbal .
- Figures 4A-C represent a map of the integrating shuttle cosmid, pYUB178, and analysis of individual clones and pools of H37Ra (pYUB178 : :H37Rv) .
- Figure 4A shows the components that allow integration of pYUB178 into the mycobacterial genomes are attP and int .
- the pYUB178 cosmid contains an E. coli ori, the L5 attP, the L5 int, a kanamycin resistance gene, aph, derived from Tn903 , lambda cos, and a unique cloning site, Sell.
- Figure 4B ia a schematic showing identification of the pYUB178/H37Rv junctional fragments within the chromosome of a H37Ra recombinant containing pYUB178 : :H37Rv D ⁇ A.
- Pstl-digested chromosomal D ⁇ A is separated by gel electrophoresis and hybridized with a labeled probe from pYUB178.
- the probe is the 1.1 kb Dral /Sspl D ⁇ A fragment of pYUB178 that flanks the Bell cloning site.
- the integrated pYUB178 : :H37Rv cosmid can be detected only by the presence of pYUB178-hybridizing DNA fragments.
- FIG. 4C are half-tones of gels showing individual H37Ra recombinants containing pYUB178 : :H37Rv cosmid clones were isolated from mouse lung tissue after spleen passage of recombinant pools, experiment J5P (see Table 9) . Pools of H37Ra (pYUB178 : :H37Rv) were collected and passaged in broth culture.
- chromosomal DNAs from pools and individual clones were isolated, digested with PstI, separated by agarose gel electrophoresis and transferred to a nylon filter to hybridize with the 1.1 kb Dral/Sspl DNA fragment of pYUB178.
- Lanes 1-3 the H37Rv DNA junctional fragments of in vivo-selected individual clones of pool 2; lanes 4 and 5, the H37Rv DNA junctional fragments of members of pool 3, before (lane 4) and after (lane 5) in vi tro passage.
- Figures 5A-B shows the growth of in vivo- selected H37Ra(pYUB178 : :H37Rv) clones in mouse lung and spleen. Growth rates of clones mc 806, H37Rv, and mc 2 816 were measured and compared. The growth rate of mc 2 806 is represented by solid squares on the solid lines, the growth rate of mc ⁇ 816 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. Figure 5A shows growth in spleen.
- Figure 5B shows growth in lung.
- Figures 6A-B illustrate the retrieval of H37Rv- containing cosmids from the mc 2 806 chromosome.
- Figure 6A is a schematic illustrating the strategy used to retrieve the H37Rv insert DNA from the integrated cosmids in H37Ra (pYUB178 : :H37Rv) recombinants.
- Figure 6B is a half-tone of an autoradiograph showing a Southern hybridization of Asel and Ec ⁇ RI digests of mc 2 806 chromosomal DNA, or cosmid DNAs that were retrieved from the chromosome of mc 806.
- the 436 bp Asel /Bell fragment of pYUB178 that contained cos was used as a probe.
- Lane 1 mc ⁇ 806 chromosomal DNA, lanes 2 to 17, DNA from sixteen individual retrieved cosmids.
- Figure 7 is a graph showing the growth of H37Ra recombinants containing pYUB352-overlapping and - nonoverlapping cosmids.
- H37Ra was separately transformed with pYUB352-overlapping cosmids, pYUB353 and pYUB354, and with unrelated cosmids, pYUB355 and pYUB356. Growth of each recombinant was measured over a time course in mouse spleen. See Table 9, experiment J36.
- the growth of pYUB353- and pYUB35 -containing H37Ra recombinants is represented by the small squares on the solid lines.
- the growth of mc 806 is represented by the large squares on the solid lines.
- FIG. 8A-C represent the restriction map of the ivg region of H37Rv DNA in pYUB352-overlapping cosmids. Restriction digests of pYUB352, pYUB353, and pYUB354 were performed with EcoRI and Hindlll.
- Figure 8A is a half-tone reproduction of gels showing digested DNA fragments which were separated by agarose gel electrophoresis.
- Figure 8B is a half-tone reproduction of gels showing DNA fragments which were hybridized to the Asel fragment of pYUB352 that included its entire H37Rv insert with flanking pYUB178 DNA sequences. The arrows point to DNA fragments that hybridize to pYUB178 DNA probes. These bands are junctional fragments. Lanes 1-3 are digests of pYUB352, lanes 4-6 are digests of pYUB353, and lanes 7-9 are digests of pYUB354. Lanes 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 Hindlll digestion patterns.
- Figure 8C is a schematic 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 H37Rv that is present in cosmids pYUB352, pYUB353, and pYUB354.
- A AseI
- E EcoRI
- H Hi_ ⁇ dIII.
- Figure 9 and 9a is comprised of four sheets .
- Figure 9 shows the nucleotide sequence of the coding strand of the 2745 bp fragment 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 Streptomyces coelicolor (GenBank Accession Nos. ⁇ 52980, ⁇ 52981, ⁇ 52983) and Streptomyces griseus (GenBank Accession 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 M. bovis ATCC35721.
- Figure 11 presents the results of a GAP comparison of Strepto_7iyces griseus principal sigma factor (Peptide translation of GenBank accession No. LO8071 from nucleotide numbers 570 to 1907, which is the coding sequence of the hrdB gene) with peptide translation of the large ORF of the approximately 3 kb DNA fragment from M. bovis associated with virulence.
- Figure 12a-l and 12a-2 (SEQ ID NO:13 and SEQ ID NO:14) is comprised of two sheets showing the large ORF of the M. bovis WAg200 sequence 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 factors of three M. tuberculosis complex strains and two Streptomyces sp.
- the present invention provides polynucleotides that are associated with virulence in members of the genus mycobacteria, and particularly in members of the mycobacterial complex. Virulence is the relative capacity of a pathogen to overcome body defenses; it is
- ISA/EP also the relative ability to cause disease in an infected host .
- virulence is
- RECTIFIED SHEET (RULE 91 ) ISA/EP generally determined by a multiplicity of traits that endow the pathogen with its ability to exploit anatomical weaknesses and overcome the immune defenses of the host . It is expected that a similar multiplicity of traits 5 determines the virulence of pathogenic mycobacteria.
- a virulent organism may be capable of killing the infected host.
- mycobacteria is meant the genus that includes the species M. phlei , M. smegmatis, M.
- tuberculosis complex including M. tuberculosis, M. bovis, M. africanum and M. microti.
- virulence factor __. encoding sequence denotes a polynucleotide sequence that encodes a product that is associated with virulence in a
- sequence associated with virulence that denotes that a polynucleotide sequence that confers a trait associated with virulence on an avirulent mycobacterium, whether or not the polynucleotide encodes a product.
- the virulence associated sequences 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 least about 70% 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 include, for example, direct comparison of sequences, and hybridization assays.
- the sequence of one of the mycobacterial DNAs associated with virulence, isolated from M. bovis, is shown in Figure 9.
- This DNA contains several contigs and an open reading frame (ORF) that based upon amino acid sequence homology in certain regions, encodes a polypeptide that is a putative sigma factor. Portions or all of fragment of which the ORF is part is in plasmids pUHAl, pUHA2, pUHA3, pUHA4, pUHA5, pUHA6, PUHA7, pUHA8, pUHA9, or pUHAll.
- a particular embodiment of the invention is an isolated or recombinant polynucleotide that is comprised of all or segment of the ORF encoding the sigma factor.
- the isolated and recombinant polynucleotides may also be comprised of sequences homologous to the mycobacterial DNA in these plasmids.
- the DNA sequences upon which the polynucleotides of the invention are based were obtained
- Cosmid genomic libraries of virulent mycobacterial strains of M. tuberculosis and M. bovis were constructed in an integrating cosmid vector.
- An example of an integrating cosmid vector is pYUB178, described by Lee et al . (1991), Proc. Natl. Acad. Sci. USA, 8.8-3111-3115 and Pascopella et al . (1994) , Infect. Immun. jS2 . :1313-1319.
- the integrating vector approximately 5 kb long, can accommodate 40-45 kb of DNA and uses the site-specific integration system of mycobacteriophage L5 to integrate recombinant DNA into a unique attB site of the mycobacterial chromosome.
- This vector thus can represent more than 95% of the entire mycobacterial genome in as few as about 300 clones.
- the recombinant DNA introduced in single copy is stably maintained in mycobacterial cells in the absence of antibiotic selection, even when the strain is passed through animals.
- use of this vector reduced the number of clones that needed to be screened, and ensured 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 include, for example, electroporation, transduction and transformation.
- the resulting libraries of recombinant clones were injected into animals, i.e., mice or guinea pigs. It is thought that clones that restore virulence may have a selective advantage and thus be enriched for
- avirulent mutants cause a self-limiting infection while virulent mycobacterial strains multiply more rapidly, and in high challenge doses cause death.
- avirulent mutants cause a self-limiting infection.
- 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 a flank, these strains are not sufficiently virulent to pass through the lymph nodes draining the injection site and enter the systemic circulation in sufficient numbers to cause gross lesions to occur in the spleen.
- the term "similar position to that present in M. bovis 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 is believed to be involved in -35 sequence recognition.
- 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 herein and factors transcribed and/or translated therefrom are associated with virulence in mycobacteria, and particularly in M. bovis or M. tuberculosis .
- polynucleotide refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers 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 modifications, for example, labels which are known in the art ( e . g. , Sambrook, et al .
- methylation substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages ( e . g. , methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) , those containing pendant moieties, such as, for example, proteins (including for e . g. , nucleases, toxins, antibodies, signal peptides, poly-L- lysine, etc.) , those with intercalators ( e . g.
- polynucleotide include both sense and antisense strands.
- Recombinant nucleic acids comprising sequences
- the wild type sequence may be employed, the wild type sequence will often be altered, e.g., by deletion, substitution, or insertion.
- the nucleic acid sequences used in this invention will usually comprise at least about 5 codons (15 nucleotides) , more usually at least about 7 to 15 codons, and most preferably at least about 35 codons. One or more introns may also be present. This number of nucleotides is usually about the minimal length required for a successful probe that would hybridize specifically with such a sequence.
- the polynucleotides of the invention will have substantial homology or similarity to the DNAs disclosed herein that are associated with virulence or with avirulence in mycobacteria.
- a nucleic acid or fragment thereof is “substantially homologous” (or “substantially similar") to another if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the 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 80%, preferably at least about 90%, and more preferably at least about 95 to 98% of the nucleotide bases.
- a nucleic acid or fragment 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 substantially more selective than total lack of specificity.
- selective hybridization 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 90%. See, Kanehisa (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 affected 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 appreciated by those skilled in the art.
- Salt concentration e.g., NaCl
- Temperatur, 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 appreciated 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. However, the combination of parameters is much more important than the measure of any single parameter. See, e.g., Wetmur and
- the polynucleotides of the invention are isolated or substantially purified.
- An "isolated” or “substantially pure” or “purified” nucleic acid is a nucleic acid, e.g., an RNA, DNA, or a mixed polymer, which is substantially separated from other mycobacterial components that naturally accompany the sequences associated with virulence, e.g., ribosomes, polymerases, and many other mycobacterial polynucleotides such as RNA and other chromosomal sequences.
- the term embraces a nucleic acid sequence 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.
- recombinant polynucleotide intends a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of a polynucleotide with which it is associated in nature; or (2) is linked to a polynucleotide other than that to which it is linked in nature; and (3) does not occur in nature.
- This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques . Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions.
- polynucleotides encode a polypeptide associated with virulence or with avirulence.
- a nucleic acid is said to
- a polypeptide if, 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 the polypeptide or a fragment thereof.
- the anti-sense strand of such a nucleic acid is also said to encode the sequence.
- 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 regulatory elements. Examples of regulatory elements usually included in expression vectors are promoters, enhancers, ribosomal binding sites, and transcription and translation initiation and termination sequences. 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 relationship with another nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.
- operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
- the regulatory elements employed in the expression vectors containing a polynucleotide encoding a virulence factor are functional in the host cell used for expression.
- 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 .
- the natural or synthetic DNA fragments coding for a desired fragment will be incorporated into recombinant nucleic
- 26 acid constructs typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell .
- DNA constructs will be suitable for autonomous replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to and integration within the genome of a cultured insect, mammalian, 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 al. (1989) or Ausubel et al. (1987 and periodic updates) .
- the polynucleotides of the present invention may also be produced by chemical synthesis, 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) J. Am. Chem. Soc. 103 :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 synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
- DNA constructs prepared for introduction into a prokaryotic or eukaryotic host will typically comprise a replication system recognized by the host, including the intended DNA fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide encoding segment.
- Expression vectors may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as
- ARS autonomously replicating sequence
- RNA splice sites ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences.
- Secretion signals from polypeptides secreted from the host cell of choice may 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 recombinant techniques well known in the art and discussed, for example, in Sambrook et. al . (1989) or Ausubel et al. (1987) .
- an appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host, and may, when appropriate, 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 e_t al. , 1987); see also, e.g., Metzger e_t al. 1988) , Nature 3_3_4:31-36. Many useful vectors are 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 enzyme promoters may be used in prokaryotic hosts. Useful yeast promoters 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 utilization, and others.
- Suitable vectors and promoters for use in yeast expression are further described in Hitzeman et. al. EP 73,657A.
- Appropriate nonnative mammalian promoters might include the early and late promoters from SV40 (Fiers et. al . . (1978) Nature 273 :113) or promoters derived from murine moloney leukemia virus, mouse mammary tumor virus, avian sarcoma
- the construct may be joined to an amplifiable gene (e.g., DHFR) so that multiple copies of the gene may be made.
- amplifiable gene e.g., DHFR
- Enhancers and Eukaryotic Gene Expression Cold Spring Harbor Press, N.Y. (1983) .
- expression vectors may replicate autonomously, they may less 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 encoding a protein necessary for 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 express 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 from complex media, e.g. the gene encoding D-alanine racemase for Bacilli.
- the choice of the proper selectable marker will depend on the host cell, and appropriate markers for different hosts are well known in the art.
- the vectors containing 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_L . , FEBS Lett. 241 :119 (1988)) , or the vectors can be introduced directly into host cells by methods well known in the art, which vary depending on the type of cellular host, including electroporation; transfection employing calcium chloride, rubidium chloride calcium phosphate, DEAE- dextran, or other substances; microprojectile
- nucleic acids and polypeptides of the present invention may be prepared by expressing the nucleic acids or portions thereof in vectors or other expression vehicles in compatible prokaryotic or eukaryotic host cells.
- prokaryotic hosts are strains of Escherichia coli, although other prokaryotes, such as Bacillus subtilis or Pseudomonas may also be used.
- Mammalian or other eukaryotic host cells such as those of yeast, filamentous fungi, plant, insect, amphibian or avian species, may also be useful for production of the proteins of the present invention. Propagation of mammalian cells in culture is per se well known.
- VERO and HeLa cells examples of commonly used mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS cell lines, although it will be appreciated by the 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 depending on the mode of the vector construction.
- the marker may 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 art, e.g., by resistance to such antibiotics as ampicillin, tetracycline.
- binding refers to an interaction or complexation between an oligonucleotide and a target nucleotide sequence, mediated through hydrogen bonding or other molecular forces.
- 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 in which adenine-thymine (or adenine-uracil) and guanine-cytosine base-pairs are formed through hydrogen bonding 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".
- 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 invention also includes recombinant host cells comprised of any of the above described polynucleotides that contain a sequence associated with virulence in mycobacteria, including those encoding a polypeptide, particularly a polypeptide that is substantially homologous to the polypeptide encoded in
- polynucleotides of the invention may be inserted into the host cell by any means known in the art, including for example, transformation, transduction, and electroporation.
- "recombinant host" any means known in the art, including for example, transformation, transduction, and electroporation.
- 31 cells refer to cells which can be, or have been, used as recipients for recombinant 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 necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
- Transformation refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, for example, direct uptake, transduction, f-mating or electroporation.
- the exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome.
- polynucleotides of the invention that are essentially homologous to sequences associated with virulence, shown in Figure 9, and in plasmids pUHAl, pUHA2, pUHA3, pUHA4, pUHA5, pUHA6, pUHA7, pUHAll and pUHA16, and in plasmids pYUB352, pYUB353, pYUB354 are of use in the detection of virulent forms of mycobacteria in biological samples.
- 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 external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, and also samples of .in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in
- oligomers of approximately 8 nucleotides or more can be prepared, either by excision from recombinant polynucleotides or synthetically, which hybridize with the mycobacterial sequences in the plasmids and are useful in identification of mycobacteria with the virulence associated trait.
- the probes for polynucleotides associated with virulence are a length which allows the detection of the virulence associated sequences by hybridization. While 6-8 nucleotides may be a workable length, sequences of 10-12 nucleotides are 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 unnecessary as the length of the fragment is increased.
- the bio ⁇ logical sample to be analyzed such as blood or serum
- the bio ⁇ logical sample to be analyzed may be treated, if desired, to extract the nucleic acids contained 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 labeling probes are known in the art, and include, for example, radioactive labels incorporated by nick translation or kinasing, biotin, fluorescent probes, and chemiluminescent probes.
- the nucleic acids extracted from the sample are then treated with the labeled probe under hybridization conditions of suitable stringencies.
- the probes can be made completely complementary
- the stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, length of time, and concentration of formamide. These factors are outlined in, for example, Maniatis, T. (1982) .
- amplification techniques in hybridization assays.
- Such techniques 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.
- PCR polymerase chain reaction
- 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 labeling may be included in the kit in separate containers.
- 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 .
- Polypeptides encoded within the sequences associated with virulence, and fragments and analogs thereof are also included as embodiments of the invention.
- the polypeptide encoded in the large ORF in Figure 9 is a putative sigma factor; thus, the intact polypeptide may exhibit the following biological activities: (1) 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
- any specific polypeptide is the binding of the polypeptide to an antibody that is directed to one or more epitopes on that polypeptide.
- the invention includes polypeptides and analogs or fragments thereof that are essentially homologous to the polypeptide encoded in the large ORF in Figure 9, and exhibit at least one of the biological activities associated with sigma factor, or alternatively, inhibits at least one of the biological activities associated with sigma factor.
- polypeptide refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as the modifications known in the art, both naturally occurring and non-naturally occurring.
- the polypeptides of the present invention will be at least about 50% homologous to the polypeptide encoded in the large ORF of Figure 9, designated herein as "virulence associated sigma factor 1" (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 conditions, to nucleic acids encoding virulence associated sigma factor 1, as well as closely related
- the length of polypeptide sequences compared for homology will generally be at least about 16 amino acids, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues.
- polypeptides when referring to polypeptides, indicates that the polypeptide or protein in question exhibits at least about 30% identity with an entire naturally occurring protein or a portion thereof, usually at least about 70% identity, and preferably at least about 95% identity.
- homology for polypeptides, is typically 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 measure of homology assigned to various substitutions, deletions, substitutions, and other modifications.
- Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
- a polypeptide "fragment,” “portion,” or “segment” is a stretch of amino acid residues of at least about 5 amino acids, often at least about 7 amino acids, typically at least about 9 to 13 amino acids, and, in various embodiments, at least about 17 or more amino acids.
- a monomeric protein is substantially pure when at least about 60 to 75% of a sample exhibits a single polypeptide sequence.
- a substantially pure protein will typically comprise about 60 to 90% W/W of a protein sample, more usually about 95%, and preferably will be over about 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel. For certain purposes higher resolution can be provided by using HPLC or other means well known in the art .
- a protein is considered to be isolated when it is separated from the contaminants which accompany it in its natural state.
- a polypeptide which is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components.
- 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 purification can be accomplished by various methods well known in the art, and include those described, e.g., in Guide to Protein Purification, ed. M. Academicr, vol. 182 of Methods in Enzvmology (Academic Press, Inc.: San Diego, 1990) and R. Scopes, Protein Purification: Principles and Practice, Springer-Verlag: New York, 1982.
- amino acid sequence of the proteins of the present invention can be determined by protein sequencing methods well known in the art.
- the present invention also provides for polypeptides or fragments thereof which are substantially
- the present invention also embraces in vivo or in vitro chemical and biochemical modifications that incorporate unusual amino acids. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labelling, e.g., with radionuclides, various enzymatic modifications, as will be readily appreciated by those well skilled in the art.
- a variety of methods for labelling polypeptides and of substituents or labels useful for such purposes are well known in the art and include radioactive isotopes such as 32 P, ligands, which bind to labeled antiligands (e.g., antibodies) , fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand.
- radioactive isotopes such as 32 P
- ligands which bind to labeled antiligands (e.g., antibodies)
- fluorophores e.g., fluorophores, chemiluminescent agents, 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 instrumentation.
- Methods of labelling polypeptides are well known in the art. See, e.g.,
- the present invention provides for fragments of the polypeptides capable of binding to antibodies directed to virulence associated sigma factor 1.
- 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.
- the present invention also 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 the virulence associated sigma factor 1 or between the sequences of the virulence associated protein and a related protein.
- heterologous fusions may be constructed which would exhibit a combination of properties or activities of the derivative proteins. See, e.g., Godowski et a_L. (1988) Science 241: 812-816.
- Fusion 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) J. Amer. Chem. Soc. 85:2149-2156.
- polypeptides of the present invention may be used in the preparation of vaccines to treat and/or prevent diseases associated with mycobacterial infections.
- Treatment refers to prophylaxis and/or therapy.
- polypeptides can be prepared as discrete entities or incorporated into a larger polypeptide, and may find use as described herein.
- the immunogenicity of the epitopes of the polypeptides of the invention may also be enhanced by preparing them in mammalian or yeast systems fused with or assembled with particle-forming proteins such as, for example, that associated with hepatitis B surface antigen. See, e.g., U.S. Pat. No. 4,722,840.
- Vaccines may be prepared from one or more im- munogenic polypeptides derived from virulence associated polypeptides, and more particularly from virulence associated sigma factor 1.
- vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solu ⁇ tion in, or suspension in, liquid prior to injection may also be prepared.
- the preparation may also be emulsified, or the protein encapsulated in liposomes.
- the active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
- the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants which may be effective include but are not limited to: aluminum 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
- N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1' - 2' -dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylam ine CGP 19835A, referred to as MTP-PE
- RIBI which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.
- the effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against an
- the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of
- suppositories and, in some cases, oral formulations or formulations suitable for distribution as aerosols.
- traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%.
- Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspen ⁇ sions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25%-70%.
- the proteins may be formulated into the vaccine as neutral or salt forms.
- Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, 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 bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
- the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective.
- the quantity to be administered which is generally in the range of 5 micrograms to 250 micrograms of antigen per dose, depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection
- Precise amounts of active ingredient required to be administered may depend on the judgment of the practitioner and may be peculiar to each subject.
- the vaccine may be given in a single dose schedule, or preferably in a multiple dose schedule.
- a multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reenforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) 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.
- the vaccine containing the im ⁇ munogenic mycobacterial antigen(s) may be administered in conjunction with other immunoregulatory agents, for example, immune globulins, as well as antibiotics.
- the immunogenic virulence associated antigens may be used for the preparation of antibodies.
- the immunogenic polypeptides prepared as described above are used to produce antibodies, including polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptide bearing an rpoV epitope (s) . Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an rpoV epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art, see for example, Mayer and Walker (1987) .
- Monoclonal antibodies directed against rpoV epitopes can also be readily produced by one skilled in
- Antibodies both monoclonal and polyclonal, which are directed against rpoV epitopes are particularly useful in diagnosis, and those which are neutralizing may be useful in passive immunotherapy.
- Monoclonal antibodies in particular, may be used to raise anti-idiotype antibodies.
- Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the antigen of the infectious agent against which protection is desired. See, for example, Nisonoff, A., et al . (1981) and Dreesman et al. (1985) . Techniques for raising anti-idiotype antibodies are known in the art. See, for example, Grzych (1985), MacNamara et al. (1984), and Uytdehaag et al . (1985) . These anti-idiotype antibodies may also be useful for treatment, vaccination and/or diagnosis of mycobacterial infections, as well as for an elucidation of the immunogenic regions of rpoV antigens.
- Both the virulence associated polypeptides and antibodies to them are useful in immunoassays to detect presence of antibodies to mycobacteria, or the presence of the virulence associated antigens, and particularly the presence of virulence associated rpoV in biological
- the immunoassay will utilize at least one epitope derived from a virulence associated polypeptide, and particularly virulence associated rpoV. In one embodiment, the immunoassay uses a combination of epitopes derived from the virulence associated polypeptide. These epitopes may be derived from the same or from different bacterial polypeptides, and may be in separate recombinant or natural polypeptides, or together in the same recombinant polypeptides.
- An immunoassay may use, for example, a monoclonal antibody directed towards a virulence associated polypeptide epitope (s) , a combina ⁇ tion of monoclonal antibodies directed towards epitopes of one mycobacterial antigen, monoclonal antibodies directed towards epitopes of different mycobacterial antigens, polyclonal antibodies directed towards the same antigen, or polyclonal antibodies 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.
- an immunoassay for an antibody(s) to a virulence associated polypeptide, and particularly to virulence associated rpoV will involve selecting and preparing the test sample suspected of containing the antibodies, such as a biological sample, then incubating it with an antigenic (i.e., epitope-containing) virulence
- the immunoassay may be, without limitations, in a heterogenous or in a homogeneous format, and of a standard or competitive type.
- the polypeptide is typically bound to a solid support to facilitate separa- tion of the sample from the polypeptide after incubation.
- 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 membranes, activated beads, and Protein A beads.
- Dynatech Immulon ⁇ - or Immulon microtiter plates or 0.25 inch polystyrene beads can be used in the heterogeneous format.
- the solid support containing the antigenic polypeptide is typically washed after separating it from the test sample, and prior to detection of bound antibod ⁇ ies. Both standard and competitive formats are known in the art .
- unlabeled anti-virulence associated polypeptide antibodies in the complex may be detected using a conjugate of antixenogeneic Ig complexed with a label, (e.g., an enzyme label) .
- a label e.g., an enzyme label
- test sample typically a biological sample
- antibodies typically a biological sample
- virulence associated polypeptide 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 components prior to testing.
- Various formats can be employed. For example, a "sandwich assay" may be employed, where antibody bound to a solid support is incubated with the test sample; washed; incubated with a second, labeled antibody to the analyte, and the support is washed again. Analyte is detected by determining if the second antibody is bound to the support.
- a test sample is usually incubated with antibody and a labeled, competing antigen is also incubated, either sequentially or simultaneously.
- an immunoassay kit comprised of one or more polypeptides of the invention, or antibodies to a polypeptide associated with virulence, and a buffer, packaged in suitable containers.
- compounds which block the activity of virulence factor associated polypeptides and particularly virulence associated rpoV may be prepared utilizing the sequence information of provided herein. This is performed by overexpressing the polypeptide, purifying the polypeptide, and then performing X-ray crystallography on the purified virulence associated polypeptide to obtain its molecular structure. Next, compounds are created which have similar molecular structures to all or portions of the polypeptide or its substrate. The compounds are then combined with the polypeptide and attached thereto so as to block one or more of its biological activities.
- the polynucleotides of the invention may also be used to produce or improve live attenuated or killed tuberculosis vaccines .
- a vaccine strain may be produced by mutating a virulence associated polynucleotide, and particularly one encoding virulence associated sigma factor 1. The mutated strain may then be formulated into a vaccine and administered to treat mycobacterial infections.
- virulence associated polynucleotides may be added to BCG vaccine strains to provide attenuated mutant tuberculosis vaccines.
- the invention also encompasses a new approach for determining factors associated with virulence or other properties of interest in other genera of bacteria 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 factor.
- Such a change could be engineered in the principal sigma factor in species of other genera of bacteria using techniques known in the art, including for example, site directed mutagenesis and homologous recombination.
- Mycobacterial species were identified by standard methods.
- tuberculosis complex strains were grown on standard mycobacterial media, harvested into buffer and inactivated by heating.
- Genomic DNA was prepared form the organisms and partially digested with a range of concentrations of Sau3AI. Fragments of 30-50 kb from these digestions were prepared using sucrose gradient centrifugation and ligated to Bell-digested pYUB178 DNA that had been treated with calf intestinal phosphatase. The ligation mixture was in vi tro -packaged into ⁇ phage heads and transduced into Escherichia coli . The kanamycin resistant recombinant clones were pooled and cosmid DNA was prepared using standard plasmid isolation methods.
- a tuberculosis complex strain of lowered virulence for guinea pigs (referred to subsequently as avirulent) was cultured in roller bottles and organisms
- pYUB178 :virulent-tuberculosis-complex-DNA.
- the electroporated organisms were plated onto media containing kanamycin and kanamycin resistant clones were pooled to form a library.
- Each member of this library had the chromosome 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 inoculated into guinea pigs. Separate guinea pigs were also inoculated with the matching avirulent tuberculosis complex strain as a control .
- virulent and avirulent strains were in the presence or absence of gross lesions in the spleen.
- the method for virulence testing in guinea pigs was adapted from the procedures described in the Trudeau Mycobacterial Culture Collection catalogue, (Anon, 1972) .
- Albino, outbred guinea pigs were inoculated subcutaneously in the flank.
- Libraries and individual strains of mycobacteria were inoculated into at least three guinea pigs which were kept in filtered-air, ventilated animal cages. Animals were sacrificed approximately 6 and 13 weeks after inoculation and examined for the presence of gross lesions of tuberculosis.
- Samples from the injection site, the prefemoral lymph nodes and spleen were cultured for mycobacteria using previously described methods.
- Formalin-fixed tissues, from the spleen, liver, kidney and lung were embedded in paraffin, sectioned at 3-5 ⁇ m, and stained with either hematoxylin and eosin (HE) or by the Ziehl-Neelsen method.
- HE hematoxylin and eosin
- a virulent M. bovis strain was isolated from bovine tissue submitted to the Wallaceville Animal
- the strain isolated from bovine tissue with the accession number 89/5276, was designated WAg200 and was cultured as described previously (Collins and de Lisle 1984) . The strain was also shown to be virulent for guinea pigs.
- Bacteriological identification 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 fragment analysis (Collins et al . 1993) .
- bacteriological identification of reisolated M. bovis strains was based on colony morphology, slow growth and growth on pyruvate- supplemented media.
- the organisms were harvested into 7 Falcon tubes each containing 50 ml phosphate buffered saline (0.14 M NaCl, 4 mM KC1, 8 mM Na 2 HP0 4 , 2 mM KH 2 P0 4 ; pH 6.5) and inactivated by heating at 75°C 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 after extraction of all organisms was 300 ⁇ g in 1 ml.
- M. bovis WAg200 DNA was partially digested with a range of concentrations of Sau3AI and digestions having the largest yield of 30-50 kb fragments were selected after analytical electrophoresis on 0.4% agarose gels
- 10 ⁇ l ligation mixture was 200 ng/ ⁇ l and the DNA molar ratio of insert to vector was 1:20.
- Four ⁇ l of the ligation mixture was in vi ro-packaged with the GigaPack
- Plasmids and M. bovis strains used in this study are listed in Tables 1 and 2.
- the receptor strain used was M. bovis ATCC35721 which had lowered virulence for guinea pigs .
- this strain is subsequently referred to as avirulent. It was inoculated into 2 x 100 ml Middlebrook 7H9 broth (Difco) containing albumin, glucose, glycerol and Tween-80 as described (Jacobs et al . 1991) .
- the cultures were grown in roller bottles at 1 revolution/min to an O.D. at 600nm of 0.18.
- the organisms were washed and concentrated to a volume of 1 ml in cold 10% glycerol and 0.4 ml were electroporated with 4 ⁇ l of pYUB178::M.
- bovis WAg200 cosmid library DNA (1 ⁇ g/ ⁇ l) as described by Jacobs et al . (1991) .
- the organisms were cultured at 37°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 1% sodium pyruvate and 10 ⁇ g/ml kanamycin.
- Approximately 4000 clones of M. bovis ATCC35721 (pYUB178 : :M. bovis WAg200) were obtained and pooled.
- a control electroporation of 400 ⁇ l organisms without added plasmid DNA yielded no kanamycin resistant colonies. Fifteen M.
- bovis ATCC35721 (pYUB178 : :M. bovis 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 fragment digestion with PstI, electrophoresis, Southern blotting and hybridization with a probe of pYUB178. This revealed
- junction fragment analysis the junction fragments of the integrated cosmid and is referred to below as junction fragment analysis. In all cases the fragment patterns were different.
- guinea pigs were inoculated subcutaneously in the flank. Libraries and individual strains of mycobacteria were inoculated into guinea pigs which were kept in filtered-air, ventilated animal cages.
- 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 M. bovis
- ATCC35721 was not sufficiently virulent to pass through the lymph nodes draining the injection site and enter the systemic circulation in sufficient numbers to cause gross lesions to occur in the spleen.
- M. bovis ATCC35721 (pYUB178 : : M. bovis WAg200) library was assessed at two time intervals and gross lesions were identified as shown in Tables 4 and 5.
- Prefemoral lymph node and spleen tissues of all guinea pigs were cultured for the presence of M. bovis .
- M. bovis organisms were isolated from all these tissues. Over 160 individual clones representing all lesion-containing prefemoral lymph nodes and spleens were subcultured and their genomic DNA subjected to junction fragment analysis. Approximately 80% of all clones had the same junction fragment pattern. Clones which gave this pattern were found in all M. bovis containing tissues. One of these ATCC35721 (pYUB178 : : M. bovis
- WAg200 WAg200 clones containing the predominant junction fragment pattern designated as WAg300 was used for further experiments below. iii . Second inoculation experiment in guinea pigs
- WAg300 and M. bovis ATCC35721 were compared concurrently.
- M. bovis strains isolated from these animals were shown to be identical to M. bovis WAg300 by junction fragment analysis.
- Genomic DNA was prepared from M. bovis WAg300, digested with the restriction enzyme NotI and ligated under conditions favoring self ligation. The ligation
- a 2 kb Aflul fragment from the insert of pUHA2 was used as a colony hybridization probe of the E. coli pYUB178:: ⁇ .. jbovis WAg200 library. Approximately one colony in every 130 library colonies gave a positive hybridization signal.
- Cosmids were isolated from 48 hybridizing clones using standard plasmid preparation methods and compared to each other and to pUHA2 on the basis of restriction enzyme digestion patterns. Three cosmids, designated pUHA3, pUHA4 and pUHA5, had most similarity to pUHA2 and are shown in Fig. 2.
- ATCC35721 (pUHA3-pUHA7) were recovered using kanamycin selection. These recombinant M. bovis clones, designated WAg301-WAg311 were inoculated into guinea pigs to assess
- bovis recombinants containing cosmids pUHA3, pUHA4, pUHA5, and pUHA7 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 pUHA3 was partially digested by Sau3AI and in separate experiments 2-4 kb and 10-15 kb fragments were cloned into the cosmid shuttle vector pUHA8.
- Vector pUHA8 was produced from pYUB178 by incorporating Pad sites on either side of the Bell cloning site.
- These libraries of pUHA3 were electroporated into M. bovis ATCC35721 to produced libraries of M. bovis ATCC35721 (pUHA8 : :pUHA3) .
- Approximately 300 colonies from the 2-4 kb library and 1000 colonies from the 10-15 kb library were pooled separately, subcultured and inoculated into guinea pigs .
- M. bovis organisms were isolated from the spleen lesions and subcultured for DNA extraction. DNA prepared from these cultures was digested with PacI and electrophoresed on
- This 3 kb sequence has sufficient overlap with the insert of pUHA2 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 of pUHA2 is within the shared DNA segment of cosmids pUHA4, pUHA5, and pUHA7.
- a restriction map of cosmid pUHA3 (Fig. 3) was constructed for the enzymes Ml ui , Nhel and NotI using a partial digestion technique.
- the cosmid insert contained no sites for the enzyme Xbal , whereas the pYUB178 vector contained two sites as shown (Fig. 3) .
- cosmid pUHA3 was partially digested with each of the three enzymes separately and then the partial digests were digested with Xbal. D ⁇ A fragments 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 with a 32 P labelled probe of the left hand vector arm of
- WAg320 was digested with Pad and the 3 kb fragment was ligated into the Pael site of the sequencing vector pUHA9 using standard methods.
- the "Erase-a-base" system Promega was used to make progressive, unidirectional deletion mutants of two clones designated pUHAll and pUHA16 which contained the 3 kb fragment in opposite orientations. Appropriately sized deletion mutants were cloned and chosen as instructed by the manufacturer's protocols. Polymerase chain reaction sequencing was performed by using commercial kits (Gibco- BRL 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.
- Figure 9A shows this sequence together with a 530 amino acid translation of the largest ORF.
- the first codon of this ORF at positions 835-837 is contiguous with the likely ribosome binding site so initiation may actually occur at codon three at positions 841-843.
- GCG Garnier-Galignment 62 package.
- An earlier version of the package is described in Devereux, J., et al . , (1984) , Nucl. Acids Res. _L2 . : 387-395.
- the comparison was performed as follows .
- the DNA sequences of the contigs were translated into amino acids (using the program TRANSLATE) and compared to the GenBank database update 82.0 using the programme TFASTA.
- This comparison revealed that the sequence analyzed had significant homology with numerous sigma factors .
- Some of the DNA sequences of the sigma factors with which the homology was particularly high were obtained from the GenBank database using the programme FETCH and their coding sequences were translated into amino acids using TRANSLATE. These sigma factors were then compared to an amino acid translation (using TRANSLATE) of the large ORF on the largest contig using the programme PILEUP.
- a smaller downstream contig was also translated using TRANSLATE and compared in the same PILEUP comparison.
- FETCH, PILEUP, TFASTA and TRANSLATE are programmes in the GCG package.
- 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 33 P. 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 fragments were isolated and sequenced and their large ORFs translated for the PILEUP comparison.
- FIG. 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) 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 effect on the translation of rpoV are shown respectively above and below the equivalent sequences from M. bovis WAg200. Two of the three mutations are also found in one or more of
- Figure 12b presents a comparison of putative principal sigma factors of four M. tuberculosis complex strains and two Streptomyces sp.
- Upper case letters denote amino acids that agree with the consensus sequence of the M. tuberculosis complex.
- An arrow denotes the position of the amino acid in the M. bovis ATCC35721 sequence that differs from that of all three of the other M. tuberculosis complex strains.
- H37Ra/H37Rv recombinants were constructed in an integrating cosmid vector, pYUB178, and electroporated into H37Ra.
- mice were infected with pools of H37Ra recombinants containing H37Rv DNA to allow the selection of growing clones in mouse spleen and lung.
- the integrating shuttle cosmid libraries based on the mycobacteriophage L5 integration system, were ideal for in vivo complementation because: (i) only approximately 225 clones were required to represent the H37Rv genome, (ii) toxic effects associated with the expression of genes from multicopy plasmids were avoided,
- the growth rates of selected recombinants were 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 identification and characterization of a 25 kb DNA fragment of M. tuberculosis which conferred an in vivo growth advantage to the growth-defective H37Ra.
- M. tuberculosis strains H37Ra and H37Rv were provided by Wilbur Jones of the Centers for Disease Control, Atlanta, and were grown in enriched 7H9 broth
- the pYUB178 integrating shuttle cosmid ( Figure 1A) was constructed by ligating the 975 bp cos-containing Bglll/Bcll fragment of lambda DNA to the
- CIP calf-intestine alkaline phosphatase
- Genomic DNA of H37Rv was prepared by mechanical disruption of bacterial cells and subsequent phenol-chloroform extractions as previously described (12) .
- H37Rv genomic DNA was partially digested with a range of concentrations of Sau3AI to generate 30-50 kb- sized fragments. Fragments of 30-50 kb were isolated as previously described (14) .
- the 30-50 kb Sau3AI fragments of chromosomal DNA were then ligated to CIP-treated, Bcll-digested pYUB178 DNA; the final DNA concentration was 50-100 ng/ ⁇ l and the DNA molar ratio of insert to vector was 1.
- the 10 -10 4 kanamycin-resistant recombinant clones were pooled and inoculated into L broth containing 25 ⁇ g/ml kanamycin.
- One aliquot was grown to prepare plasmid DNA by an alkaline 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 v 2764 transductants containing the pYUB178 : :H37Rv library was approximately 1 x 10 9 cfu/ml.
- the lysate was stored at 4°C after filtering through a 0.45 ⁇ m pore sterile filter.
- H37Ra (pYUB178 : :H37Rv) recombinant pools.
- H37Ra culture was electroporated with the pYUB178 : :H37Rv library DNA in plasmid form, and separately, with pYUB178 DNA. Approximately 450 transformants arose from five independent electroporations of cells with approximately 1 ⁇ g library DNA each.
- Two pools of H37Ra (pYUB178 : :H37Rv) recombinants, pool 1 and pool 2 were made by collecting and inoculating approximately 225 colonies into 50 ml of enriched 7H9 broth containing 10 ⁇ g/ml kanamycin, and allowing growth for approximately two weeks . Aliquots of pools were distributed and frozen in cryovials for later use in animal experiments.
- pool 3 Another pool of H37Ra (pYUB178 : :H37Rv) recombinants, pool 3, consisted of approximately 260 clones and was used to determine whether the pools were representative. Recombinants of pool 3 were collected directly from plates of enriched Middlebrook 7H10 agar
- mice were inoculated with each recombinant group or control group per timepoint. Inoculation of mice with spleen-passaged bacteria was accomplished 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 filtered through sterile gauze to exclude tissue clumps.
- the filtrate 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 7H9 broth for subsequent mouse experiments and DNA analyses.
- H37Ra (pYUB178 : :H37Rv) recombinant clones using chemical disruption of bacterial cells as previously described
- DNA was partially digested with Sau3Al; fragments of 30-50 kb were size-fractionated and eluted from
- the 30-50 kb fragments were ligated to the 975 bp Bglll/Bcll fragment containing cos of coliphage lambda DNA.
- the ligation conditions were such that the final DNA concentration was 50 to 100 ng/ ⁇ l, and the molar ratio of chromosomal DNA fragments to cos DNA fragments was 1.
- the ligation mixture was packaged into lambda phage heads and tails using the Stratagene GigaPack kit, and transduced into E. coli strain HBlOl. Individual kanamycin-resistant transductant colonies were picked and cosmid DNA was isolated. Cosmid DNA was then analyzed by restriction digestion and Southern hybridization.
- Probes were labeled with ⁇ - P ⁇ dCTP using random hexamer priming with the Pharmacia oligolabeling kit (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) , or with horseradish peroxidase according to the protocol of the Enhanced Chemiluminescence ECL Gene Detection System (Amersham International, Amersham, UK) .
- H37Ra transformant colonies from each of the four electroporations were grown in enriched 7H9 broth containing kanamycin to prepare sufficient culture for mouse experiments.
- the in vivo growth rates of H37Ra containing pYUB352-overlapping and -nonoverlapping clones were measured in the experiment designated J36 (see Table
- the integrating cosmid pYUB178 contains an E. coli ori derived from pUC19, the L5 attP site, the L5 integrase gene, a kanamycin resistance gene, aph, derived from Tn903 , the lambda cos sequence and a unique cloning site, Bell (see Figure 4A) .
- the L5 mycobacteriophage contains an E. coli ori derived from pUC19, the L5 attP site, the L5 integrase gene, a kanamycin resistance gene, aph, derived from Tn903 , the lambda cos sequence and a unique cloning site, Bell (see Figure 4A) .
- the H37Rv library was constructed by ligating 40 kb size-selected chromosomal DNA fragments, generated by partial digestion with Sau3AI , to alkaline
- mice were intravenously infected with either
- H37Ra (pYUB178 : :H37Rv) recombinant pool 1 or 2.
- mouse spleens were individually homogenized, pooled, and used to infect a second group of mice. Individual recombinant colonies that grew from the plated lung homogenates prepared from the second group of mice were picked.
- chromosomal DNAs were isolated from these individual recombinants and subjected to Southern analysis with a pYUB178 probe.
- the junctional fragment analyses of selected individual recombinants from the in vivo-passed pool 2 in experiment J5P are shown in Figure 4C, lanes 1, 2 and 3.
- Lane 1 shows the clone designated mc ⁇ 807
- lane 2 shows the clone
- lane 3 shows a clone that has junctional fragments identical to those of mc 2 806. Because clones having junctional fragments identical to those of mc 806 were isolated from many animals during two different experiments, J2P and J5P, (data not shown) , mc 2 806 was further characterized.
- Clone mc 2 806 did not grow faster than mc 2 816 during the first two weeks in mouse lung ( Figure 5B) . Therefore the faster in vivo growth rate of mc 2 806 compared to mc' ⁇ ie was evident only in mouse spleen. The growth rates of mc 806, mc 2 816, and H37Rv in enriched 7H9 broth were virtually identical (data not shown) .
- H37Rv DNA insert present in an in vivo-selected recombinant was responsible for its in vivo growth phenotype, it had to be retrieved from the chromosome.
- a disadvantage of the stably integrating pYUB178 : :H37Rv cosmid library is the difficulty of cosmid retrieval from the chromosome of a H37Ra (pYUB178 : :H37Rv) recombinant; the excision functions of L5 are not yet understood.
- a method was devised to clone the
- H37Rv DNA insert as a cosmid (see Figure 6A) .
- H37Rv insert DNA was responsible for the spleen growth phenotype, it had to be reintroduced into H37Ra and tested. Reintroduction of the H37Rv insert DNA from the mc 2 806 recombinant into
- H37Ra required a replicating vector. Retrieved cosmids did not have the ability to replicate in mycobacteria because they lost the int gene when they were removed from the chromosomes of the recombinants. Therefore, pYUB352 DNA was used as a probe to screen the pYUB178: :H37RV library in E. coli for the H37Rv DNA insert associated with mc 806. Colonies of E. coli
- H37Rv DNA of mc 2 806 confers in vivo growth advantage to H37Ra
- inocula were estimated from cfu retained in the spleen on day 1; spleen retention is usually 10% of the inoculating dose.
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US20188094A | 1994-02-24 | 1994-02-24 | |
US201880 | 1994-02-24 | ||
US26557994A | 1994-06-24 | 1994-06-24 | |
US265579 | 1994-06-24 | ||
US29269594A | 1994-08-18 | 1994-08-18 | |
US292695 | 1994-08-18 | ||
PCT/US1994/014912 WO1995017511A2 (en) | 1993-12-23 | 1994-12-23 | Mycobacteria virulence factors and a method for their identification |
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US5700683A (en) * | 1995-02-17 | 1997-12-23 | Pathogenesis Corporation | Virulence-attenuating genetic deletions deleted from mycobacterium BCG |
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ES2313725T3 (en) * | 1995-05-30 | 2009-03-01 | Astrazeneca Ab | NEW DNA MOLECULES. |
US6444444B1 (en) | 1996-07-10 | 2002-09-03 | Aventis Pasteur Limited | Genes encoding mycobacterial proteins associated with cell binding and cell entry and uses thereof |
US6221364B1 (en) * | 1996-11-12 | 2001-04-24 | Albert Einstein College Of Medicine Of Yeshiva University | Recombinant mycobacteria auxotrophic for diaminopimelate |
WO1998032862A2 (en) * | 1997-01-29 | 1998-07-30 | Leopold Flohe | L-alanine dehydrogenase of mycobacterium marinum |
US6613881B1 (en) * | 1997-05-20 | 2003-09-02 | Corixa Corporation | Compounds for immunotherapy and diagnosis of tuberculosis and methods of their use |
US6136324A (en) | 1997-08-21 | 2000-10-24 | Connaught Laboratories Limited | Attenuated strains of mycobacteria |
WO2001059071A2 (en) | 2000-02-09 | 2001-08-16 | Genvec, Inc. | Methods of preparing and using a viral vector library |
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