U.S. patent application number 09/754153 was filed with the patent office on 2001-09-06 for dna molecule fragments encoding for cellular uptake of mycobacterium tuberculosis and uses thereof.
Invention is credited to Chong, Pele, Riley, Lee W..
Application Number | 20010019716 09/754153 |
Document ID | / |
Family ID | 24768338 |
Filed Date | 2001-09-06 |
United States Patent
Application |
20010019716 |
Kind Code |
A1 |
Riley, Lee W. ; et
al. |
September 6, 2001 |
DNA molecule fragments encoding for cellular uptake of
mycobacterium tuberculosis and uses thereof
Abstract
The present invention relates to a DNA molecule conferring on
Mycobacterium tuberculosis an ability to enter mammalian cells and
to survive within macrophages. Peptides, proteins, or polypeptides
(e.g. the Mycobacterium cell entry protein or Mcep) encoded by this
gene fragment are useful in vaccines to prevent infection by
Mycobacterium tuberculosis, while the antibodies raised against
these peptides, proteins, or polypeptides can be employed in
passively immunizing those already infected by the organism. These
proteins, peptides, polypeptides, and antibodies may be utilized in
diagnostic assays to detect Mycobacterium tuberculosis in tissue or
bodily fluids. The peptides, proteins, or polypeptides of the
present invention can be associated with various other therapeutic
materials, for administration to mammals, particularly humans, to
achieve uptake of those materials by such cells. Synthetically
constructed peptides based on the disclosed amino acid sequences
exhibit the same mammalian cell uptake activity observed with
Mcep.
Inventors: |
Riley, Lee W.; (New York,
NY) ; Chong, Pele; (Richmond Hill, CA) |
Correspondence
Address: |
Michael L. Goldman, Esq.
NIXON PEABODY LLP
Clinton Square
P. O. Box 31051
Rochester
NY
14603
US
|
Family ID: |
24768338 |
Appl. No.: |
09/754153 |
Filed: |
January 4, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09754153 |
Jan 4, 2001 |
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08689411 |
Aug 7, 1996 |
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6224881 |
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Current U.S.
Class: |
424/190.1 ;
435/183; 536/23.2 |
Current CPC
Class: |
A61P 37/04 20180101;
A61K 48/00 20130101; A61P 31/06 20180101; A61K 39/00 20130101; C07K
14/35 20130101; A61K 38/00 20130101 |
Class at
Publication: |
424/190.1 ;
536/23.2; 435/183 |
International
Class: |
C07H 021/04; A61K
039/04 |
Claims
What is claimed:
1. An isolated DNA molecule conferring an ability to enter
mammalian cells, wherein said DNA molecule is a fragment of the DNA
molecule in Mycobacterium tuberculosis which confers on that
organism an ability to enter mammalian cells.
2. An isolated DNA molecule according to claim 1, wherein said DNA
molecule encodes the amino acid sequence corresponding to SEQ. ID.
No. 8 or SEQ. ID. No. 10.
3. An isolated DNA molecule according to claim 2, wherein said DNA
molecule corresponds to the nucleotide sequence corresponding to
SEQ. ID. No. 7 or SEQ. ID. No. 9.
4. An isolated DNA molecule according to claim 1, wherein said DNA
molecule encodes a variant of the amino acid sequence corresponding
to SEQ. ID. No. 8 or SEQ. ID. No. 10 with the variant being
unchanged at its amino acid positions corresponding to the
following amino acids in SEQ. ID. No. 4: positions 27 and 28,
position 32, or position 38.
5. An isolated peptide encoded by a DNA molecule according to claim
1.
6. An isolated peptide according to claim 5, wherein the protein or
polypeptide has an amino acid sequence corresponding to SEQ. ID.
No. 8 or SEQ. ID. No. 10.
7. An isolated peptide according to claim 5, wherein said DNA
molecule has a nucleotide sequence corresponding to SEQ. ID. No. 7
or SEQ. ID. No. 9.
8. An isolated peptide according to claim 5, wherein the peptide is
a variant of the amino acid corresponding to SEQ. ID. No. 8 or SEQ.
ID. No. 10 with the variant being unchanged at its amino acid
positions corresponding to the following amino acids in SEQ. ID.
No. 4: positions 27 and 28, position 32, or position 38.
9. An isolated peptide according to claim 5, wherein said peptide
is recombinant.
10. An isolated peptide according to claim 5, wherein said peptide
is purified.
11. A method of vaccinating mammals against infection by
Mycobacterium tuberculosis comprising: administering an effective
amount of the isolated peptide according to claim 5 to mammals.
12. A method according claim 11 wherein said administering is oral,
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, or intranasal.
13. A recombinant DNA expression system comprising an expression
vector into which is inserted a heterologous DNA according to claim
1.
14. A recombinant DNA expression system according claim 13, wherein
said heterologous DNA corresponds to the nucleotide sequence
corresponding to SEQ. ID. No. 7 or SEQ. ID. No. 9.
15. A recombinant DNA expression system according to claim 13,
wherein said heterologous DNA is inserted into said vector in
proper orientation and correct reading frame.
16. A host cell incorporating a heterologous DNA according to claim
1.
17. A host cell according to claim 16, wherein said heterologous
DNA corresponds to the nucleotide sequence corresponding to SEQ.
ID. No. 7 or SEQ. ID. No. 9.
18. A host cell according to claim 16, wherein said heterologous
DNA is inserted in a recombinant DNA expression system comprising
an expression vector.
19. A vaccine for preventing infection and disease of mammals by
Mycobacterium tuberculosis comprising: an isolated peptide
according to claim 5; and a pharmaceutically-acceptable
carrier.
20. A vaccine according to claim 19, wherein said peptide is
purified.
21. A method of vaccinating mammals against infection by
Mycobacterium tuberculosis comprising: administering an effective
amount of the vaccine according to claim 19 to mammals.
22. A method according claim 21, wherein said administering is
oral, intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, or intranasal.
23. An isolated antibody or binding portion thereof or a probe
against a peptide according to claim 5.
24. An isolated antibody or binding portion thereof or probe
according to claim 23, wherein said antibody is monoclonal or
polyclonal.
25. An isolated antibody or binding portion thereof or probe
according to claim 23, wherein said antibody is specific for an
antigenic determinant of said peptide encoded by a gene fragment
conferring on Mycobacterium tuberculosis an ability to enter
mammalian cells.
26. A method of passively immunizing mammals infected with
Mycobacterium tuberculosis comprising: administering an effective
amount of said antibody or binding portion thereof or probe
according to claim 23 to mammals infected with Mycobacterium
tuberculosis.
27. A method according to claim 26, wherein said administering is
oral, intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, or intranasal.
28. A composition for passively immunizing mammals infected with
Mycobacterium tuberculosis comprising: an isolated antibody or
binding portion thereof or probe according to claim 23; and a
pharmaceutically-acceptable carrier.
29. A composition according to claim 28, wherein said antibody is
monoclonal or polyclonal.
30. A composition according to claim 28, wherein said antibody is
specific for an antigenic determinant of said peptide encoded by a
gene fragment conferring on Mycobacterium tuberculosis an ability
to enter mammalian cells.
31. A method of passively immunizing mammals infected with
Mycobacterium tuberculosis comprising: administering an effective
amount of said composition according to claim 28 to mammals
infected with Mycobacterium tuberculosis.
32. A method according to claim 31, wherein said administering is.
oral, intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, or intranasal.
33. A method for detection of Mycobacterium tuberculosis in a
sample of tissue or body fluids comprising: providing a peptide
according to claim 5 as an antigen; contacting the sample with the
antigen; and detecting any reaction which indicates that
Mycobacterium tuberculosis is present in the sample using an assay
system.
34. A method according to claim 33, wherein the assay system is
selected from the group consisting of an enzyme-linked
immunosorbent assay, a radioimmunoassay, a gel diffusion precipitin
reaction assay, an immunodiffusion assay, an agglutination assay, a
fluorescent immunoassay, a protein A immunoassay, and an
immunoelectrophoresis assay.
35. A method for detection of Mycobacterium tuberculosis in a
sample of tissue or body fluids comprising: providing an antibody
or binding portion thereof or probe according to claim 23;
contacting the sample with the antibody or binding portion thereof
or probe; and detecting any reaction which indicates that
Mycobacterium tuberculosis is present in the sample using an assay
system.
36. A method according to claim 35, wherein the assay system is
selected from the group consisting of an enzyme-linked
immunosorbent assay, a radioimmunoassay, a gel diffusion precipitin
reaction assay, an immunodiffusion assay, an agglutination assay, a
fluorescent immunoassay, a protein A immunoassay, and an
immunoelectrophoresis assay.
37. A method for detection of Mycobacterium tuberculosis in a
sample of tissue or body fluids comprising: providing a nucleotide
sequence of the DNA molecule according to claim 1 as a probe in a
nucleic acid hybridization assay; contacting the sample with the
probe; and detecting any reaction which indicates that
Mycobacterium tuberculosis is present in the sample.
38. A method for detection of Mycobacterium tuberculosis in a
sample of tissue or body fluids comprising: providing a nucleotide
sequence of the DNA molecule according to claim 1 as a probe in a
gene amplification detection procedure; contacting the sample with
the probe; and detecting any reaction which indicates that
Mycobacterium tuberculosis is present in the sample.
39. A product for uptake of materials into mammalian cells
comprising: a material for uptake by mammalian cells; and a peptide
according to claim 5, wherein said peptide is associated with said
material.
40. A product according to claim 39, wherein said peptide has an
amino acid sequence corresponding to SEQ. ID. No. 8.
41. A product according to claim 39, wherein said peptide has an
amino acid sequence corresponding to SEQ. ID. No. 10.
42. A product according to claim 39, wherein the peptide is a
variant of the amino acid corresponding to SEQ. ID. No. 8 or SEQ.
ID. No. 10 with the variant being unchanged at its amino acid
positions corresponding to the following amino acids in SEQ. ID.
No. 4: positions 27 and 28, position 32, or position 38.
43. A product according to claim 39, wherein said peptide is
purified.
44. A product according to claim 39, wherein said material is
selected from the group consisting of antibiotics, DNA fragments,
anti-neoplastic agents, and mixtures thereof.
45. A cellular uptake process comprising: directing a material into
mammalian cells with a peptide according to claim 5.
46. A process according to claim 45, wherein said peptide has an
amino acid sequence corresponding to SEQ. ID. No. 8.
47. A process according to claim 45, wherein said peptide has an
amino acid sequence corresponding to SEQ. ID. No. 10.
48. A process according to claim 45, wherein the peptide is a
variant of the amino acid corresponding to SEQ. ID. No. 8 or SEQ.
ID. No. 10 with the variant being unchanged at its amino acid
positions corresponding to the following amino acids in SEQ. ID.
No. 4: positions 27 and 28, position 32, or position 38.
49. A process according to claim 45, wherein said peptide is
purified.
50. A process according to claim 45, wherein said material is
selected from the group consisting of antibiotics, DNA fragments,
anti-neoplastic agents, and mixtures thereof.
51. A product for uptake of materials into mammalian cells
comprising: a material for uptake by mammalian cells; and a peptide
according to claim 5, wherein said peptide is associated with said
material.
52. A cellular uptake process comprising: directing a material into
mammalian cells with a peptide according to claim 5.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a DNA molecule encoding for
uptake of Mycobacterium tuberculosis and its use in drugs,
vaccines, and diagnostic tests.
BACKGROUND OF THE INVENTION
[0002] Tuberculosis is the leading cause of death in the world with
an estimated 9 million new cases of tuberculosis and 2.9 million
deaths occurring from the disease each year. In the United States,
the steadily declining incidents of tuberculosis has been reversed
since 1985. This problem is compounded by the increasing incidence
of drug-resistant strains of Mycobacterium tuberculosis.
[0003] Recent outbreaks of tuberculosis have involved settings in
which a large number of HIV-infected persons resided in close
proximity (e.g., AIDS wards in hospitals, correctional facilities,
and hospices). Transmission of tuberculosis to health care workers
occurred in these outbreaks; 18 to 50% of such workers showed a
conversion in their skin tests. See F. Laraque et. al.,
"Tuberculosis in HIV-Infected Patients," The AIDS Reader
(September/October 1992), which is hereby incorporated by
reference.
[0004] There are two basic clinical patterns that follow infection
with Mycobacterium tuberculosis.
[0005] In the majority of cases, inhaled tubercle bacilli ingested
by phagocytic alveolar macrophages are either directly killed or
grow intracellularly to a limited extent in local lesions called
tubercles. Infrequently in children and immunocompromised
individuals, there is early hematogenous dissemination with the
formation of small miliary (millet-like) lesions or
life-threatening meningitis. More commonly, within 2 to 6 weeks
after infection, cell-mediated immunity develops, and infiltration
into the lesion of immune lymphocytes and activated macrophages
results in the killing of most bacilli and the walling-off of this
primary infection, often without symptoms being noted by the
infected individual. Skin-test reactivity to a purified protein
derivative ("PPD") of tuberculin and, in some cases, X-ray evidence
of a healed, calcified lesion provide the only evidence of the
infection. Nevertheless, to an unknown extent, dormant but viable
Mycobacterium tuberculosis bacilli persist.
[0006] The second pattern is the progression or breakdown of
infection to active disease. Individuals infected with
Mycobacterium tuberculosis have a 10% lifetime risk of developing
the disease. In either case, the bacilli spread from the site of
initial infection in the lung through the lymphatics or blood to
other parts of the body, the apex of the lung and the regional
lymph node being favored sites. Extrapulmonary tuberculosis of the
pleura, lymphatics, bone, genito-urinary system, meninges,
peritoneum, or skin occurs in about 15% of tuberculosis patients.
Although many bacilli are killed, a large proportion of
infiltrating phagocytes and lung parenchymal cells die as well,
producing characteristic solid caseous (cheese-like) necrosis in
which bacilli may survive but not flourish. If a protective immune
response dominates, the lesion may be arrested, albeit with some
residual damage to the lung or other tissue. If the necrotic
reaction expands, breaking into a bronchus, a cavity is produced in
the lung, allowing large numbers of bacilli to spread with coughing
to the outside. In the worst case, the solid necrosis, perhaps a
result of released hydrolases from inflammatory cells, may liquefy,
which creates a rich medium for the proliferation of bacilli,
perhaps reaching 10.sup.9 per milliliter. The pathologic and
inflammatory processes produce the characteristic weakness, fever,
chest pain, cough, and, when a blood vessel is eroded, bloody
sputum.
[0007] Ignorance of the molecular basis of virulence and
pathogenesis is great. It has been suggested that the establishment
of molecular evidence regarding avirulent strains, the
identification and cloning of putative virulence genes of the
pathogen, and the demonstration that virulence can be conveyed to
an avirulent strain by those genes is necessary. Although avirulent
strains of Mycobacterium tuberculosis exist, the nature of the
mutations is unknown. Not a single gene involved in the
pathogenesis of tuberculosis has been defined in the prior art. The
molecular bases of invasion of host cells, intracellular survival,
growth, spread, or tissue tropism also have not been known. None of
the targets of existing drugs has been characterized at a molecular
level, and the mechanism of resistance to any drug has not been
defined; no new mycobacterial target for drug development has been
characterized in 20 years.
[0008] There have been many prescribed treatment regimens for
tuberculosis. The regimen recommended by the U.S. Public Health
Service and the American Thoracic Society is a combination of
isoniazid, rifampicin, and pyrazinamide for two months followed by
administration of isoniazid and rifampicin for an additional four
months. In persons with HIV infection, isoniazid and rifampicin
treatment are continued for an additional seven months. This
treatment, called the short-course chemotherapy, produces a cure
rate of over 90% for patients who complete it. Treatment for
multi-drug resistant tuberculosis requires addition of ethambutol
and/or streptomycin in the initial regimen, or second line drugs,
such as kanamycin, amikacin, capreomycin, ethionamide,
cyclcoserine, PAS, and clofazimine. New drugs, such as
ciprofloxacin and ofloxacin can also be used. For individuals
infected with conventional Mycobacterium tuberculosis and showing
PPD positive results, chemoprophylaxis with isoniazid has been
about 90% effective in preventing the disease. Tuberculosis and
these treatments are discussed in more detail in B. Bloom et. al.,
"Tuberculosis: Commentary on a Reemergent Killer," Science,
257:1055-64 (1992); "Control of Tuberculosis in the United States,"
American Thoracic Society, 146:1623-33 (1992); City Health
Information, vol. 11 (1992), which is hereby incorporated by
reference.
[0009] Although the currently used treatments for tuberculosis have
a relatively high level of success, the need remains to improve the
success rate for treating this disease. Moreover, in view of the
ever-increasing level of Mycobacterium tuberculosis strains which
are resistant to conventional treatment regimens, new types of
treatment must be developed. In high tuberculosis endemic areas,
both in the United States and abroad, such resistant strains are
becoming increasingly present.
SUMMARY OF THE INVENTION
[0010] The present invention relates to isolated DNA molecules
conferring on Mycobacterium tuberculosis an ability to enter
mammalian cells and/or to survive within macrophages as well as
isolated peptides, proteins, or polypeptides encoded by those
isolated DNA molecules. Of particular interest are DNA molecules
conferring an ability to enter mammalian cells, wherein the DNA
molecules are fragments of the DNA molecule in Mycobacterium
tuberculosis which confers on that organism an ability to enter
mammalian cells. Also of interest are the corresponding encoded
peptides, proteins, or polypeptides. The DNA molecules can be
inserted as heterologous DNA in an expression vector forming a
recombinant DNA expression system for producing the peptides,
proteins, or polypeptides. Likewise, the heterologous DNA, usually
inserted in an expression vector to form a recombinant DNA
expression system, can be incorporated in a cell to achieve this
objective.
[0011] The isolated peptides, proteins, or polypeptides of the
present invention can be combined with a
pharmaceutically-acceptable carrier to form a vaccine or used alone
for administration to mammals, particularly humans, for preventing
infection by Mycobacterium tuberculosis. Alternatively, each of the
peptides, proteins, or polypeptides of the present invention can be
used to raise an antibody, a binding portion thereof, or probe. The
antibody, binding portion thereof, or probe may be used alone or
combined with a pharmaceutically-acceptable carrier to treat
mammals, particularly humans, already exposed to Mycobacterium
tuberculosis to induce a passive immunity to prevent disease
occurrence.
[0012] The peptides, proteins, or polypeptides of the present
invention or the antibodies or binding portions thereof raised
against them (as well as probes) can also be utilized in a method
for detection of Mycobacterium tuberculosis in a sample of tissue
or body fluids. When the peptides, proteins, or polypeptides are
utilized, they are provided as an antigen. Any reaction with the
antigen or the antibody is detected using an assay system which
indicates the presence of Mycobacterium tuberculosis in the sample.
Alternatively, Mycobacterium tuberculosis can be detected in such a
sample by providing a nucleotide sequence of the gene conferring on
Mycobacterium tuberculosis an ability to enter mammalian cells
and/or to survive within macrophages or a fragment thereof as a
probe in a nucleic acid hybridization assay or a gene amplication
detection procedure (e.g., using a polymerase chain reaction
procedure) . Any reaction with the probe is detected so that the
presence of Mycobacterium tuberculosis in the sample is
indicated.
[0013] The peptides, proteins, or polypeptides of the present
invention can also be used for purposes unrelated to the treatment
or detection of Mycobacterium tuberculosis. More particularly, the
ability of those peptides, proteins, or polypeptides to confer on
Mycobacterium tuberculosis an ability to enter mammalian cells can
be utilized to permit such cells to uptake other materials. This
can be achieved with a product that includes a material for uptake
by mammalian cells and the peptides, proteins, or polypeptides of
the present invention associated or bonded (e.g., covalently
linked) with that material.
[0014] Isolation of the DNA molecules of the present invention
constitutes a significant advance in the treatment and detection of
such bacteria. It also provides the basis for a vaccine to prevent
infection by Mycobacterium tuberculosis and a pharmaceutical agent
for passive immunization for those exposed to Mycobacterium
tuberculosis. The peptides, proteins, or polypeptides utilized in
the vaccine or to produce the pharmaceutical agent can be produced
at high levels using recombinant DNA technology.
[0015] In diagnostic applications, the peptides, proteins, or
polypeptides of the present invention as well as antibodies and
binding portions thereof against them permit rapid determination of
whether a particular individual is infected with Mycobacterium
tuberculosis. Moreover, such detection can be carried out without
requiring an examination of the individual being tested for an
antibody response.
[0016] Aside from the development of treatments and diagnostic
tools for Mycobacterium tuberculosis, the present invention's
ability to confer entry of such organisms into mammalian cells has
significant utility in therapeutic treatments requiring the
introduction of materials into cells, particularly to macrophages.
By associating the peptide, protein, or polypeptide of the present
invention with pharmaceutical agents, such agents can be rapidly
introduced into cells for treatment thereof. The enhanced cellular
uptake of such products can reduce drug dosages, thus reducing
toxicity and cost. For example, in conventional cancer treatment,
drug toxicity is a major problem due to the requirement for
administration of large dosages; the present invention has the
potential to reduce such high dosage levels while enabling delivery
of equivalent or higher drug levels intracellularly.
[0017] Furthermore, binding the peptides, proteins, or polypeptides
of the present invention to DNA fragments can be utilized in
conjunction with gene therapy regimens. In particular, the ability
of the encoded product of the DNA molecules of the present
invention to augment uptake into macrophages provides an
opportunity to deliver genes specifically to macrophages. Such a
system can be used to induce not only humoral immunity but
cell-mediated immunity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A, 1B, and 1C are thin-section electron micrographs
of HeLa cells infected with Mycobacterium tuberculosis strain,
including H37Ra (ATCC25177) (FIG. 1A), and the invasive recombinant
strain E. coli XL1-Blue (pZX7) (FIG. 1B and 1C). An
electron-transparent zone surrounds the Mycobacterium tuberculosis
organism (arrow in FIG. 1A). The cells were incubated with
Mycobacterium tuberculosis strain for 72 hours in FIG. 1A and with
XL1-Blue (pZX7) for 7.5 hours in FIGS. 1B and 1C. Multiple
organisms are visible in FIG. 1C, suggesting bacterial
proliferation inside phagosomes. The bars represent 0.5 .mu.m.
[0019] FIG. 2 shows the construction of unidirectional deletional
subclones (pZX7.3, pZX7.4, pZX7.5, and pZX7.6) and Bam HI-Pst I
(pZX7.1), Pst I-HinD III (pZX7.2), and Bam HI-Eco RI (pZX7.7)
subclones from the original vector pZX7. The black bars represent
the Mycobacterium tuberculosis DNA sequences, and the white bars
represent pBluescript sequences. The subclone vectors were
transferred into E. coli XL1-Blue and then incubated with these
transformed strains for 6 hours with a HeLa cell monolayer.
[0020] FIGS. 3A, 3B, and 3C are thin-section electron micrographs
of human macrophages exposed to the invasive recombinant E. coli
clone XL1-Blue(pZX7) for 3 hours (FIG. 3A) and. 24 hours (FIG. 3B)
compared with cells exposed to nonpathogenic E. coli XL1-Blue
(pBluescript) for 24 hours (FIG. 3C). The bacteria become
compartmentalized, surrounded by layers of membrane inside the
macrophage (FIG. 3B). No bacteria were visible after 24 hours by
electron microscopy in macrophages exposed to
XL1-Blue(pBluescript). The bars represent 1 .mu.m.
[0021] FIG. 4 shows the SDS-polyacrylamide gel electrophoresis of
an acetone-precipitated soluble fraction of bacterial cell
sonicate. The polypeptides were analyzed in a 9% gel (left):
molecular size standards (lane 1), E. coli XL1-Blue with a vector
(pZN7) containing an unrelated Mycobacterium tuberculosis DNA
fragment between the Bam HI-Eco RI pBluescript cloning sites (lane
2), and XL1-Blue(pZX7) (lane 3). Analysis in an 8% gel (right):
XL1-Blue containing a vector (pZX7.8) with a two-base frameshift
introduced 12 bases upstream from the Bam HI cloning site in pZX7
(lane 1) and XL1-Blue(pZX7) (lane 2). Molecular sizes are indicated
at the far-right. We detected a 52-kD polypeptide in the soluble
protein fraction of XL1-Blue(pZX7) (arrow). A protein of about 50
kD is expressed by XL1-Blue containing pZX7.8. The expression of
the 52-kD protein was always associated with HeLa cell interaction
of the recombinant E. coli clone.
[0022] FIG. 5 shows an SDS-PAGE analysis of recombinant E. coli
lysates with the low molecular weight marker in lane 1, E. coli
BL21(DE3) in lane 2, E.coli BL21(DE3)(pET23c) in lane 3, E. coli
BL21(DE3)(pET23c-ORF1), uninduced in lane 4, and E. coli BL21(DE3)
(pET23c-ORF1) induced in lane 5.
[0023] FIGS. 6A and B show a transmission electron microscopy study
of the association of latex beads coated with Mycobacterium
tuberculosis invasion-association recombinant protein (i.e. Mcep)
with HeLa cells. FIG. 6A shows recombinant protein-coated beads
(arrow). FIG. 6B shows control E. coli lysate protein-coated beads
(arrow).
[0024] FIG. 7 is a transmission electron microscopy showing the
uptake of 0.3 .mu.m latex microspheres coated with a 22-amino acid
peptide Inv3 derived from the Mycobacterium tuberculosis
invasion-association recombinant protein (i.e. Mcep) after
incubation for 4 hours.
DETAILED DESCRIPTION OF THE INVENTION
[0025] One aspect of the present invention relates to an isolated
DNA molecule conferring on Mycobacterium tuberculosis an ability to
enter mammalian cells and to survive within macrophages. This DNA
molecule comprises the nucleotide sequence corresponding to SEQ.
ID. No. 1 as follows:
1 GGATCGAATT GCTGGCCTTT GGCGGGCGAT TCGTGGAGAT CGCCCGTAGA AAGGTTCGCG
60 GACGCCAAGG CCGCCGCAGA CCGCCATAAA CGTAGTTGAC CAGGTGGTCT
TGACTGGGGC 120 CGGACACCGA CGTGAACGAG GCGACCCGAT CCGCGTTACA
TCCACCTGAT TCCGGCAAAT 180 GTGAACGCCG ACATCAAGGC GACCACGGTG
TTCGGCGGTA AGTATGTGTC GTTGACCACG 240 CCGAAAAACC CGACAAAGAG
GCGGATAACG CCAAAAGACG TCATCGACGT ACGGTCGGTG 300
[0026] The above DNA molecule encodes for a polypeptide having a
molecular weight of about 50 to 55 kilodaltons, preferably 52
kilodaltons. The amino acid sequence, deduced from the nucleotide
sequence corresponding to SEQ. ID. No. 1, represents a highly
hydrophilic protein with a hydrophobic region at its carboxy
terminus. It could be a secreted protein, a cytoplasmic protein, or
a surface protein with its carboxy terminus attached to the outer
membrane of the organism. It is believed that this protein or
polypeptide has the deduced amino acid sequence corresponding to
SEQ. ID. No. 2 as follows:
2 Gly Ser Asn Cys Trp Pro Leu Ala Gly Asp Ser Trp Arg Ser Pro Val 1
5 10 15 Glu Arg Phe Ala Asp Ala Lys Ala Ala Ala Asp Arg His Lys Arg
Ser 20 25 30 Xaa Pro Gly Gly Leu Asp Trp Gly Arg Thr Pro Thr Xaa
Thr Arg Arg 35 40 45 Pro Asp Pro Arg Tyr Ile His Leu Ile Pro Ala
Asn Val Asn Ala Asp 50 55 60 Ile Lys Ala Thr Thr Val Phe Gly Gly
Lys Tyr Val Ser Leu Thr Thr 65 70 75 80 Pro Lys Asn Pro Thr Lys Arg
Arg Ile Thr Pro Lys Asp Val Ile Asp 85 90 95 Val Arg Ser Val Thr
Thr Glu Ile Asn Thr Leu Phe Gln Thr Leu Thr 100 105 110 Ser Ile Ala
Glu Lys Val Asp Pro Val Lys Leu Asn Leu Thr Leu Ser 115 120 125 Ala
Ala Ala Glu Ala Leu Thr Gly Leu Gly Asp Lys Phe Gly Glu Ser 130 135
140 Ile Val Asn Ala Asn Thr Val Leu Asp Asp Leu Asn Ser Arg Met Pro
145 150 155 160 Gln Ser Arg His Asp Ile Gln Gln Leu Ala Ala Leu Gly
Asp Val Tyr 165 170 175 Ala Asp Ala Ala Pro Asp Leu Phe Asp Phe Leu
Asp Ser Ser Val Thr 180 185 190 Thr Ala Arg Thr Ile Asn Ala Gln Gln
Ala Glu Leu Asp Ser Ala Leu 195 200 205 Leu Ala Ala Ala Gly Phe Gly
Asn Thr Thr Ala Asp Val Phe Asp Arg 210 215 220 Gly Gly Pro Tyr Leu
Gln Arg Gly Val Ala Asp Leu Val Pro Thr Ala 225 230 235 240 Thr Leu
Leu Asp Thr Tyr Ser Pro Glu Leu Phe Cys Thr Ile Arg Asn 245 250 255
Phe Tyr Asp Ala Asp Arg Pro Asp Arg Gly Ala Ala Ala Xaa Ala Arg 260
265 270 Ser Gly Ser Arg Ser Ala Arg Arg Thr Ser Lys Xaa Phe Ala Pro
Phe 275 280 285 Phe Ala His Leu Pro Ala Ala Val Asp Val Ser Thr Arg
Gln Ala Ala 290 295 300 Glu Ala Asp Leu Ala Gly Lys Ala Ala Gln Tyr
Arg Pro Asp Glu Leu 305 310 315 320 Ala Arg Tyr Ala Gln Arg Val Met
Asp Trp Leu His Pro Asp Gly Asp 325 330 335 Leu Thr Asp Thr Glu Arg
Ala Arg Lys Arg Gly Ile Thr Leu Ser Asn 340 345 350 Gln Gln Tyr Asp
Gly Met Ser Arg Leu Ser Gly Tyr Leu Thr Pro Gln 355 360 365 Ala Arg
Ala Thr Phe Glu Ala Val Leu Ala Lys Leu Ala Ala Pro Gly 370 375 380
Ala Thr Asn Pro Asp Asp His Thr Pro Val Ile Asp Thr Thr Pro Asp 385
390 395 400 Ala Ala Ala Ile Asp Arg Asp Thr Arg Ser Gln Ala Gln Arg
Asn His 405 410 415 Asp Gly Leu Leu Ala Gly Leu Arg Ala Leu Ile Arg
His Pro Ala Ile 420 425 430 Ser Ala Leu Gly Ala Ala Asn Ser Arg Cys
Cys Ala Val His Ala Glu 435 440 445 Arg Met His Ala Ile Ser Asn Trp
Leu Ala Pro Tyr Ser Gly Trp Asn 450 455 460 Cys Ser Ile Ala Met Pro
Ala Ala Val Ala Ala Ala Leu Thr Ser Arg 465 470 475 480 Thr Asn Ala
Ser Cys Ser Ser Thr Pro Ala Thr Pro Tyr Cys Ala His 485 490 495 Ser
Val Glu Gly Ser Arg Trp Pro Ser Ala Ser Thr Lys Arg Asn 500 505
510
[0027] In the immediately-preceding sequence, Xaa signifies a stop
codon. Production of this isolated protein or polypeptide is
preferably carried out using recombinant DNA technology. The
protein or polypeptide is believed to have one or more antigenic
determinants conferring on Mycobacterium tuberculosis an ability to
enter mammalian cells and to survive within macrophages.
[0028] As indicated by the presence of the stop codons in above
SEQ. ID. Nos. 1 and 2, these sequences constitute or are encoded by
several open reading frames. The first open reading frame extends
from position 181 to 5 position 807 of the nucleotide sequence of
SEQ. ID. No. 1. This sequence which confers an ability to enter
mammalian cells has the following nucleotide sequence (SEQ. ID. No.
3):
3 GTGAACGCCG ACATCAAGGC GACCACGGTG TTCGGCGGTA AGTATGTGTC GTTGACCACG
60 CCGAAAAACC CGACAAAGAG GCGGATAACG CCAAAAGACG TCATCGACGT
ACGGTCGGTG 120 ACCACCGAGA TCAACACGTT GTTCCAGACG CTCACCTCGA
TCGCCGAGAA GGTGGATCCG 180 GTCAAGCTGA ACCTGACCCT GAGCGCGGCC
GCGGAGGCGT TGACCGGGCT GGGCGATAAG 240 TTCGGCGAGT CGATCGTCAA
CGCCAACACC GTTCTGGATG ACCTCAATTC GCGGATGCCG 300 CAGTCGCGCC
ACGACATTCA GCAATTGGCG GCTCTGGGCG ACGTCTACGC CGACGCGGCG 360
CCGGACCTGT TCGACTTTCT CGACAGTTCG GTGACCACCG CCCGCACCAT CAATGCCCAG
420 CAAGCGGAAC TGGATTCGGC GCTGTTGGCG GCGGCCGGGT TCGGCAACAC
CACAGCCGAT 480 GTCTTCGACC GCGGCGGGCC GTATCTGCAG CGGGGGGTCG
CCGACCTGGT CCCCACCGCC 540 ACCCTGCTCG ACACTTATAG CCCGGAACTG
TTCTGCACGA TCCGCAACTT CTACGATGCC 600 GATCGACCTG ACCGCGGGGC TGCCGCA
627
[0029] The nucleotide sequence corresponding to SEQ. ID. No. 3
encodes for the following amino acid sequence (SEQ. ID. No. 4):
4 Val Asn Ala Asp Ile Lys Ala Thr Thr Val Phe Gly Gly Lys Tyr Val 1
5 10 15 Ser Leu Thr Thr Pro Lys Asn Pro Thr Lys Arg Arg Ile Thr Pro
Lys 20 25 30 Asp Val Ile Asp Val Arg Ser Val Thr Thr Glu Ile Asn
Thr Leu Phe 35 40 45 Gln Thr Leu Thr Ser Ile Ala Glu Lys Val Asp
Pro Val Lys Leu Asn 50 55 60 Leu Thr Leu Ser Ala Ala Ala Glu Ala
Leu Thr Gly Leu Gly Asp Lys 65 70 75 80 Phe Gly Glu Ser Ile Val Asn
Ala Asn Thr Val Leu Asp Asp Leu Asn 85 90 95 Ser Arg Met Pro Gln
Ser Arg His Asp Ile Gln Gln Leu Ala Ala Leu 100 105 110 Gly Asp Val
Tyr Ala Asp Ala Ala Pro Asp Leu Phe Asp Phe Leu Asp 115 120 125 Ser
Ser Val Thr Thr Ala Arg Thr Ile Asn Ala Gln Gln Ala Glu Leu 130 135
140 Asp Ser Ala Leu Leu Ala Ala Ala Gly Phe Gly Asn Thr Thr Ala Asp
145 150 155 160 Val Phe Asp Arg Gly Gly Pro Tyr Leu Gln Arg Gly Val
Ala Asp Leu 165 170 175 Val Pro Thr Ala Thr Leu Leu Asp Thr Tyr Ser
Pro Glu Leu Phe Cys 180 185 190 Thr Ile Arg Asn Phe Tyr Asp Ala Asp
Arg Pro Asp Arg Gly Ala Ala 195 200 205 Ala
[0030] The protein or polypeptide encoded by this amino acid
sequence has one or more antigenic determinants conferring on
Mycobacterium tuberculosis an ability to enter mammalian cells.
This protein or polypeptide has a molecular weight of 23-28
kilodaltons, preferably 25 kilodaltons.
[0031] The sequences corresponding to SEQ. ID. Nos. 1 and 2 contain
or are encoded by an additional open reading frame which is
believed to confer on Mycobacterium tuberculosis an ability to
survive within macrophages. The nucleotide sequence corresponding
to this open reading frame is as follows (SEQ. ID. No. 5):
5 GTGGATGTGT CCACCCGCCA GGCCGCCGAA GCCGACCTGG CCGGCAAAGC CGCTCAATAT
60 CGTCCCGACG AGCTGGCCCG CTACGCCCAG CGGGTCATGG ACTGGCTACA
CCCCGACGGC 120 GACCTCACCG ACACCGAACG CGCCCGCAAA CGCGGCATCA
CCCTGAGCAA CCAGCAATAC 180 GACGGCATGT CACGGCTAAG TGGCTACCTG
ACCCCCCAAG CGCGGGCCAC CTTTGAAGCC 240 GTGCTAGCCA AACTGGCCGC
CCCCGGCGCG ACCAACCCCG ACGACCACAC CCCGGTCATC 300 GACACCACCC
CCGATGCGGC CGCCATCGAC CGCGACACCC GCAGCCAAGC CCAACGCAAC 360
CACGACGGGC TGCTGGCCGG GCTGCGCGCG CTGATCCGTC ATCCTGCCAT CTCGGCCCTC
420 GGCGCCGCCA ACTCCAGGTG CTGTGCGGTC CACGCCGAAC GCATGCACGC
GATCTCGAAT 480 TGGTTGGCAC CGTATTCGGG ATGGAACTGC TCGATAGCGA
TGCCTGCTGC CGTTGCCGCG 540 GCGTTGACAT CGCGGACGAA CGCCTCGTGC
TCGAGCACCC CGGCGACACC GTACTGCGCC 600 CACAGCGTCG AAGGCAGCCG
CTGGCCGTCC GCGTCGACCA AGAGGAATTC 650
[0032] The nucleotide sequence corresponding to SEQ. ID. No. 5
encodes for a protein or polypeptide having the following amino
acid sequence (SEQ. ID. No. 6):
6 Val Asp Val Ser Thr Arg Gln Ala Ala Glu Ala Asp Leu Ala Gly Lys 1
5 10 15 Ala Ala Gln Tyr Arg Pro Asp Glu Leu Ala Arg Tyr Ala Gln Arg
Val 20 25 30 Met Asp Trp Leu His Pro Asp Gly Asp Leu Thr Asp Thr
Glu Arg Ala 35 40 45 Arg Lys Arg Gly Ile Thr Leu Ser Asn Gln Gln
Tyr Asp Gly Met Ser 50 55 60 Arg Leu Ser Gly Tyr Leu Thr Pro Gln
Ala Arg Ala Thr Phe Glu Ala 65 70 75 80 Val Leu Ala Lys Leu Ala Ala
Pro Gly Ala Thr Asn Pro Asp Asp His 85 90 95 Thr Pro Val Ile Asp
Thr Thr Pro Asp Ala Ala Ala Ile Asp Arg Asp 100 105 110 Thr Arg Ser
Gln Ala Gln Arg Asn His Asp Gly Leu Leu Ala Gly Leu 115 120 125 Arg
Ala Leu Ile Arg His Pro Ala Ile Ser Ala Leu Gly Ala Ala Asn 130 135
140 Ser Arg Cys Cys Ala Val His Ala Glu Arg Met His Ala Ile Ser Asn
145 150 155 160 Trp Leu Ala Pro Tyr Ser Gly Trp Asn Cys Ser Ile Ala
Met Pro Ala 165 170 175 Ala Val Ala Ala Ala Leu Thr Ser Arg Thr Asn
Ala Ser Cys Ser Ser 180 185 190 Thr Pro Ala Thr Pro Tyr Cys Ala His
Ser Val Glu Gly Ser Arg Trp 195 200 205 Pro Ser Ala Ser Thr Lys Arg
Asn 210 215
[0033] The protein or polypeptide conferring on Mycobacterium
tuberculosis an ability to survive within macrophages has a
molecular weight of at least 21 kilodaltons. It is expected that in
nature this protein or polypeptide has a weight greater than the 21
kilodaltons of SEQ. ID. No. 6, because SEQ. ID. No. 6 is encoded by
a DNA molecule with no stop codon at its terminus. See SEQ. ID. No.
5. Therefore, in nature, the protein or polypeptide conferring
survival within macrophages is believed to be longer.
[0034] Also encompassed by the present invention are fragments of
the above DNA molecules and the proteins or polypeptides they
encode. Suitable fragments are constructed by using appropriate
restriction sites, revealed by inspection of the DNA molecule's
sequence, to: (i) insert an interposon (Felly, et al., "Interposon
Mutagenesis of Soil and Water Bacteria: A Family of DNA Fragments
Designed for in vitro Insertion Mutagenesis of Gram-negative
Bacteria," +E,uns Gene 52:147-15 (1987), which is hereby
incorporated by reference) such that truncated forms of the
polypeptides or proteins of the present invention, that lack
various amounts of the C-terminus, can be produced or (ii) delete
various internal portions of the protein. Alternatively, the
sequence can be used to amplify any portion of the coding region,
such that it can be cloned into a vector supplying both
transcription and translation start signals. Of particular interest
are fragments of the DNA molecule which confers on Mycobacterium
tuberculosis an ability to enter mammalian cells (i.e. fragments of
SEQ. ID. No. 3) and the encoded protein or polypeptide (i.e.
fragments of SEQ. ID. No. 4).
[0035] One example of such a DNA molecule fragment is defined by
the nucleotide sequence correspondong to SEQ. ID. No. 7 as
follows:
7 GTGAACGCCG ACATCAAGGC GACCACGGTG TTCGGCGGTA AGTATGTGTC GTTGACCACG
60 CCGAAAAACC CGACAAAGAG GCGGATAACG CCAAAAGACG TCATCGACGT
ACGGTCGGTG 120 ACCACCGAGA TCAACACGTT GTTCCAGACG CTCACCTCGA
TCGCCGAGAA GGTGGATCCG 180
[0036] The nucleotide sequence corresponding to SEQ. ID. No. 7
encodes for a peptide having the following amino acid (SEQ. ID. No.
8):
8 Val Asn Ala Asp Ile Lys Ala Thr Thr Val Phe Gly Gly Lys Tyr Val 1
5 10 15 Ser Leu Thr Thr Pro Lys Asn Pro Thr Lys Arg Arg Ile Thr Pro
Lys 20 25 30 Asp Val Ile Asp Val Arg Ser Val Thr Thr Glu Ile Asn
Thr Leu Phe 35 40 45 Gln Thr Leu Thr Ser Ile Ala Glu Lys Val Asp
Pro 50 55 60
[0037] This amino acid sequence encompasses 60 amino acids at the
entire N-terminus of the protein or polypeptide corresponding to
SEQ. ID. No. 4. When latex microspheres are coated with this
peptide, the coated spheres are taken up by HeLa cells to the same
extent as beads coated with the whole protein of SEQ. ID. No.
4.
[0038] Another example of DNA molecule fragments of the DNA
molecule which confers on Mycobacterium tuberculosis an ability to
enter mammalian cells (i.e. SEQ. ID. No. 3) is defined by the
nucleotide sequence corresponding to SEQ. ID. No. 9 as follows:
9 ACAAAGAGGC GGATAACGCC AAAAGACGTC ATCGACGTAC GGTCGGTGAC CACCGAGATC
60 AACACG 66
[0039] The nucleotide sequence corresponding to SEQ. ID. No. 9
encodes for a peptide having the following amino acid (SEQ. ID. No.
10):
10 Thr Lys Arg Arg Ile Thr Pro Lys Asp Val Ile Asp Val Arg Ser Val
1 5 10 15 Thr Thr Glu Ile Asn Thr 20
[0040] This amino acid sequence encompasses the amino acids from
positions 25 to 46 of the amino acid sequence corresponding to SEQ.
ID. No. 4. This peptide, when coated on latex microspheres, at a
nanomolar concentration was sufficient to permit uptake of the
spheres by Hela cells.
[0041] Variants may also (or alternatively) be made by, for
example, the deletion or addition of amino acids that have minimal
influence on the properties, secondary structure and hydropathic
nature of the polypeptide. For example, a polypeptide may be
conjugated to a signal (or leader) sequence at the N-terminal end
of the protein which co-translationally or post-translationally
directs transfer of the protein. The polypeptide may also be
conjugated to a linker or other sequence for ease of synthesis,
purification, or identification of the polypeptide. Further, as
demonstrated infra in Example 12, variants of the polypeptides
having amino acids corresponding to SEQ. ID. Nos. 8 and 10 can be
utilized provided that the variant is unchanged at its amino acid
positions corresponding to the following amino acids in SEQ. ID.
No. 4: positions 27 and 28, position 32, or position 38.
[0042] In addition, it may be advantageous to modify the peptides
in order to impose a conformational restraint upon them. This might
be useful, for example, to mimic a naturally-occurring conformation
of the peptide in the context of the native protein in order to
optimize the effector immune responses that are elicited.
[0043] Modified peptides are referred to herein as "peptide
analogs". The term "peptide analog" extends to any functional
chemical equivalent of a peptide characterized by its increased
stability and/or efficacy and immunogenicity +E,uns in vivo or
+E,uns in vitro in respect of the practice of the invention. The
term "peptide analog" is also used herein to extent any amino acid
derivative of the peptides as described herein. Peptide analogs
contemplated herein are produced by procedures that include, but
are not limited to, modifications to side chains, incorporation of
unnatural amino acids and/or their derivatives during peptide
synthesis and the use of cross-linkers and other methods which
impose conformational constraint on the peptides or their
analogs.
[0044] It will be apparent that the peptides employed herein as
antigens can be modified in a variety of different ways without
significantly affecting the functionally important immunogenic
behaviour thereof. Possible modifications to the peptide sequence
may include the following:
[0045] One or more individual amino acids can be substituted by
amino acids having comparable or similar properties, thus:
[0046] V may be substituted by I;
[0047] T may be substituted by S;
[0048] K may be substituted by R; or
[0049] L may be sustituted by I, V, or M.
[0050] One or more of the amino acids of the peptides of the
invention can be replaced by a "retro-inverso" amino acid, i.e., a
bifunctional amine having a functional group corresponding to an
amino acid, as discussed in published International application WO
91/13909, which is hereby incorporated by reference.
[0051] One or more amino acids can be deleted.
[0052] Structural analogs mimicking the 3-dimensional structure of
the peptide can be used in place of the peptide.
[0053] Examples of side chain modifications contemplated by the
present invention include modification of amino groups, such as by
reductive alkylation by reaction with an aldehyde followed by
reduction with NaBH; amidation with methylacetimidate; acetylation
with acetic anhydride; carbamylation of amino groups with 2, 4, 6,
trinitrobenzene sulfonic acid (TNBS); alkylation of amino groups
with succinic anhydride and tetrahydrophthalic anhydride; and
pyridoxylation of lysine with pyridoxal-5'-phosphate followed by
reduction with NaBH.sub.4.
[0054] The guanidino group of arginine residues may be modified by
the formation of heterocyclic condensation products with reagents,
such as 2,3-butanedione, phenylglyoxal and glyoxal.
[0055] The carboxyl group may be modified by carbodiimide
activation via o-acylisourea formation followed by subsequent
derivatisation, for example, to a corresponding amide.
[0056] Sulfhydryl groups may be modified by methods, such as
carboxymethylation with iodoacetic acid or iodoacetamide; performic
acid oxidation to cysteic acid; formation of mised disulphides with
other thiol compounds, reaction with maleimide; maleic anhydride or
other substituted maeimide; formation of mercurial derivatives
using 4-chloromercuribenzoate, 4-chloromercuriphenylsulfonic acid,
phenylmercury chloride, 2-chloromercuric-4-nitrophenol and other
mercurials; carbamylation with cyanate at alkaline pH.
[0057] Tryptophan residues may be modified by, for example,
oxidation with N-bromosuccinimide or alkylation of the indole ring
with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides. Tyrosine
residues may be altered by nitration with tetranitromethane for
form a 3-nitrotyrosine derivative.
[0058] Modification of the imidazole ring of a histidine residue
may be accomplished by alkylation with iodoacetic acid derivatives
or N-carbethoxylation with diethylpyrocarbonate.
[0059] Examples of incorporating unnatural amino acids and
derivatives during peptide synthesis include, but are not limited
to, use of norleucine, 4-amino butyric acid,
4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid,
t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine,
4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or
D-isomers of amino acids.
[0060] Further, the peptides of the present invention may be
lipidated with, for example, cholesterol or palmitate to
incorporate it into cationic liposomes.
[0061] The peptides, proteins, or polypeptides of the present
invention are preferably produced in purified form by conventional
techniques. For instance, see Examples 5-6 infra. To isolate the
proteins, the E. coli host cell carrying a recombinant plasmid is
propagated, homogenized, and the homogenate is centrifuged to
remove bacterial debris. The supernantant is then subjected to
sequential ammonium sulfate precipitation. The fraction containing
the proteins of the present invention are subjected to gel
filtration in an appropriately sized dextran or polyacrylamide
column to separate the proteins. If necessary, the protein fraction
may be further purified by other chromatography, such as by
HPLC.
[0062] Any one of the DNA molecules conferring on Mycobacterium
tuberculosis an ability to enter mammalian cells and/or to survive
within macrophages can be incorporated in cells using conventional
recombinant DNA technology. Generally, this involves inserting the
selected DNA molecule into an expression system to which that DNA
molecule is heterologous (i.e. not normally present). The
heterologous DNA molecule is inserted into the expression system or
vector in proper orientation and correct reading frame. The vector
contains the necessary elements for the transcription and
translation of the inserted protein-coding sequences.
[0063] U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby
incorporated by reference, describes the production of expression
systems in the form of recombinant plasmids using restriction
enzyme cleavage and ligation with DNA ligase. These recombinant
plasmids are then introduced by means of transformation and
replicated in unicellular cultures including procaryotic organisms
and eucaryotic cells grown in tissue culture.
[0064] Recombinant genes may also be introduced into viruses, such
as vaccina virus. Recombinant viruses can be generated by
transfection of plasmids into cells infected with virus.
[0065] Suitable vectors include, but are not limited to, the
following viral vectors such as lambda vector system gt11, gt
WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325,
pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37,
pKC101, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene
Cloning Systems" Catalog (1993) from Stratagene, La Jolla, Calif,
which is hereby incorporated by reference), pQE, pIH821, pGEX, pET
series (see F. W. Studier et. al., "Use of T7 RNA Polymerase to
Direct Expression of Cloned Genes," +E,uns Gene Expression
Technology vol. 185 (1990), which is hereby incorporated by
reference) and any derivatives thereof. Recombinant molecules can
be introduced into cells via transformation, particularly
transduction, conjugation, mobilization, or electroporation. The
DNA sequences are cloned into the vector using standard cloning
procedures in the art, as described by Maniatis et al., Molecular
Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs
Harbor, New York (1982), which is hereby incorporated by
reference.
[0066] A variety of host-vector systems may be utilized to express
the protein-encoding sequence(s). Primarily, the vector system must
be compatible with the host cell used. Host-vector systems include
but are not limited to the following: bacteria transformed with
bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such
as yeast containing yeast vectors; mammalian cell systems infected
with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell
systems infected with virus (e.g., baculovirus). The expression
elements of these vectors vary in their strength and specificities.
Depending upon the host-vector system utilized, any one of a number
of suitable transcription and translation elements can be used.
[0067] Different genetic signals and processing events control many
levels of gene expression (e.g., DNA transcription and messenger
RNA (mRNA) translation).
[0068] Transcription of DNA is dependent upon the presence of a
promotor which is a DNA sequence that directs the binding of RNA
polymerase and thereby promotes mRNA synthesis. The DNA sequences
of eucaryotic promotors differ from those of procaryotic promoters.
Furthermore, eucaryotic promotors and accompanying genetic signals
may not be recognized in or may not function in a procaryotic
system, and, further, procaryotic promoters are not recognized and
do not function in eucaryotic cells.
[0069] Similarly, translation of mRNA in procaryotes depends upon
the presence of the proper procaryotic signals which differ from
those of eucaryotes. Efficient translation of mRNA in procaryotes
requires a ribosome binding site called the Shine-Dalgarno (SD)
sequence on the mRNA. This sequence is a short nucleotide sequence
of mRNA that is located before the start codon, usually AUG, which
encodes the amino-terminal methionine of the protein. The SD
sequences are complementary to the 3'-end of the 16S rRNA
(ribosomal RNA) and probably promote binding of mRNA to ribosomes
by duplexing with the rRNA to allow correct positioning of the
ribosome. For a review on maximizing gene expression, see Roberts
and Lauer, Methods in Enzymology, 68:473 (1979), which is hereby
incorporated by reference.
[0070] Promotors vary in their "strength" (i.e. their ability to
promote transcription). For the purposes of expressing a cloned
gene, it is desirable to use strong promoters in order to obtain a
high level of transcription and, hence, expression of the gene.
Depending upon the host cell system utilized, any one of a number
of suitable promoters may be used. For instance, when cloning in E.
coli, its bacteriophages, or plasmids, promotors such as the T7
phage promoter, lac promotor, trp promotor, recA promotor,
ribosomal RNA promotor, the P.sub.R and P.sub.L promoters of
coliphage lambda and others, including but not limited, to lacUV5,
ompF, bla, lpp, and the like, may be used to direct high levels of
transcription of adjacent DNA segments. Additionally, a hybrid
trp-lacUV5 (tac) promotor or other E. coli promoters produced by
recombinant DNA or other synthetic DNA techniques may be used to
provide for transcription of the inserted gene.
[0071] Bacterial host cell strains and expression vectors may be
chosen which inhibit the action of the promotor unless specifically
induced. In certain operons, the addition of specific inducers is
necessary for efficient transcription of the inserted DNA. For
example, the lac operon is induced by the addition of lactose or
IPTG (isopropylthio-beta-D-galac- toside). A variety of other
operons, such as trp, pro, etc., are under different controls.
[0072] Specific initiation signals are also required for efficient
gene transcription and translation in procaryotic cells. These
transcription and translation initiation signals may vary in
"strength" as measured by the quantity of gene specific messenger
RNA and protein synthesized, respectively. The DNA expression
vector, which contains a promotor, may also contain any combination
of various "strong" transcription and/or translation initiation
signals. For instance, efficient translation in E. coli requires a
Shine-Dalgarno (SD) sequence about 7-9 bases 5' to the initiation
codon (ATG) to provide a ribosome binding site. Thus, any SD-ATG
combination that can be utilized by host cell ribosomes may be
employed. Additionally, any SD-ATG combination produced by
recombinant DNA or other techniques involving incorporation of
synthetic nucleotides may be used.
[0073] Once the desired isolated DNA molecule conferring on
Mycobacterium tuberculosis an ability to enter mammalian cells
and/or to survive within macrophages has been cloned into an
expression system, it is ready to be incorporated into a host cell.
Such incorporation can be carried out by the various forms of
transformation noted above, depending upon the vector/host cell
system. Suitable host cells include, but are not limited to,
bacteria, virus, yeast, mammalian cells, and the like. Peptides can
also be constructed synthetically as an alternative to recombinant
formation. For example, the peptides corresponding to SEQ. ID. Nos.
8 and 10 were prepared by a peptide synthesizer.
[0074] Generally, the human immune system responds to infection by
pathogenic bacteria by producing antibodies that bind to specific
proteins or carbohydrates on the bacterial surface. The antibodies
stimulate binding to macrophages which have receptors that bind to
the F.sub.c region of the antibodies. Other serum proteins, called
complement, coat the foreign particle and stimulate their ingestion
by binding to specific surface receptors on the macrophage. Once
the particle is bound to the surface of the macrophage, the
sequential process of ingestion begins by continual apposition of a
segment of the plasma membrane to the particle surface. Surface
receptors on the membranes then interact with ligands distributed
uniformity over the particle surface to link the surfaces together.
The macrophage enveloping the particle is then delivered to
lysosomes where the particle is ingested.
[0075] Some organisms are ingested (i.e. undergo uptake) by
macrophages but are not killed. Amongst these is Mycobacterium
tuberculosis. As a result, such organisms are able to survive
indefinitely within macrophages and, when they escape from the
macrophage, cause active tuberculosis.
[0076] In view of the present invention's determination of
nucleotide sequences conferring on Mycobacterium tuberculosis an
ability to enter mammalian cells, the molecular basis for
Mycobacterium tuberculosis uptake is suggested. With this
information and the above-described recombinant DNA technology, a
wide array of therapeutic and/or prophylatic agents and diagnostic
procedures for, respectively, treating and detecting Mycobacterium
tuberculosis can be developed.
[0077] For example, an effective amount of the peptides, proteins,
or polypeptides of the present invention can be administered alone
or in combination with a pharmaceutically-acceptable carrier to
humans, as a vaccine, for preventing infection by Mycobacterium
tuberculosis. Alternatively, it is possible to administer to
individuals exposed to Mycobacterium tuberculosis an effective
amount of an antibody or binding portion thereof against these
peptides, proteins, or polypeptides or probes as a passive
immunization. Such antibodies or binding portions thereof or probes
are administered alone or in combination with a
pharmaceutically-acceptable carrier to effect short term treatment
of individuals who may have been recently exposed to Mycobacterium
tuberculosis.
[0078] Antibodies suitable for use in inducing passive immunity can
be monoclonal or polyclonal.
[0079] Monoclonal antibody production may be effected by techniques
which are well-known in the art. Basically, the process involves
first obtaining immune cells (lymphocytes) from the spleen of a
mammal (e.g., mouse) which has been previously immunized with the
antigen of interest (i.e. the peptide, protein, or polypeptide of
the present invention) either in vivo or in vitro. The
antibody-secreting lymphocytes are then fused with (mouse) myeloma
cells or transformed cells, which are capable of replicating
indefinitely in cell culture, thereby producing an immortal,
immunoglobulin-secreting cell line. The resulting fused cells, or
hybridomas, are cultured and the resulting colonies screened for
the production of the desired monoclonal antibodies. Colonies
producing such antibodies are cloned, and grown either in vivo or
in vitro to produce large quantities of antibody. A description of
the theoretical basis and practical methodology of fusing such
cells is set forth in Kohler and Milstein, Nature 256:495 (1975),
which is hereby incorporated by reference.
[0080] Mammalian lymphocytes are immunized by in vivo immunization
of the animal (e.g., a mouse) with one of the peptides, proteins,
or polypeptides of the present invention. Such immunizations are
repeated as necessary at intervals of up to several weeks to obtain
a sufficient titer of antibodies. The virus is carried in
appropriate solutions or adjuvants. Following the last antigen
boost, the animals are sacrificed and spleen cells removed.
[0081] Fusion with mammalian myeloma cells or other fusion partners
capable of replicating indefinitely in cell culture is effected by
standard and well-known techniques, for example, by using
polyethylene glycol (PEG) or other fusing agents (See Milstein and
Kohler, Eur. J. Immunol. 6:511 (1976), which is hereby incorporated
by reference). This immortal cell line, which is preferably murine,
but may also be derived from cells of other mammalian species,
including but not limited to rats and humans, is selected to be
deficient in enzymes necessary for the utilization of certain
nutrients, to be capable of rapid growth and to have good fusion
capability. Many such cell lines are known to those skilled in the
art, and others are regularly described.
[0082] Procedures for raising polyclonal antibodies are also well
known. Typically, such antibodies can be raised by administering
one of the peptides, proteins, or polypeptides of the present
invention subcutaneously to New Zealand white rabbits which have
first been bled to obtain pre-immune serum. The antigens can be
injected at a total volume of 100 .mu.l per site at six different
sites. Each injected material will contain synthetic surfactant
adjuvant pluronic polyols, or pulverized acrylamide gel containing
the protein or polypeptide after SDS-polyacrylamide gel
electrophoresis. The rabbits are then bled two weeks after the
first injection and periodically boosted with the same antigen
three times every six weeks. A sample of serum is then collected 10
days after each boost. Polyclonal antibodies are then recovered
from the serum by affinity chromatography using the corresponding
antigen to capture the antibody. Ultimately, the rabbits are
euthenized with pentobarbitol 150 mg/Kg IV. This and other
procedures for raising polyclonal antibodies are disclosed in E.
Harlow, et. al., editors; Antibodies: A Laboratory Manual (1988),
which is hereby incorporated by reference. For instance, see
Example 9 infra.
[0083] As indicated, both polyclonal and monoclonal antibodies may
be employed in accordance with the present invention. Of special
interest to the present invention are antibodies which are produced
in humans or are "humanized" (i.e., non-immunogenic in a human) by
recombinant or other technology. Humanized antibodies may be
produced, for example, by placing an immunogenic portion of an
antibody with a corresponding, but non-immunogenic portion,
chimeric antibodies. See, for example, International Patent
Publication No. PCT/US86/02269 to Robinson et al.; European Patent
Application No. 184,187 to Akira et al.; European Patent
Application No. 171,496 to Taniguchi, M.; European Patent
Application No. 173,494 to Morrison et al.; PCT Application No.
W086/01533 to Neuberger et al.; European Patent Application No.
125,023 to Cabilly et al.; Better et al., Science 240:1041-43
(1988); Liu et al., PNAS 84:3439-43 (1987); Lie et al., J. Immunol.
139:3521-26 (1987); Sun et al., PNAS 84:214-18 (1987); Nishimura et
al., Cancer Research 47:999-05 (1987); Wood et al., Nature
314:446-49 (1985); and Shaw et al., J. National Cancer Inst.
80:1553-59 (1988), all of which are hereby incorporated by
reference. General reviews of "humanized" chimeric antibodies are
provided by Morrison, S. L., Science 229:1202-07 (1985) and Oi et
al., Bio Techniques 4:214 (1986), which are hereby incorporated by
reference.
[0084] In addition to utilizing whole antibodies, the present
invention encompasses use of binding portions of such antibodies.
Such binding portions include Fab fragments, F(ab') .sub.2
fragments, and Fv fragments. Such antibody fragments can be made by
conventional procedures, such as proteolytic fragmentation
procedures, as described in J. Goding, Monoclonal Antibodies:
Principles and Practice, pp. 98-118 (N.Y. Academic press 1983),
which is hereby incorporated by reference.
[0085] Alternatively, the processes of the present invention can
utilize probes found either in nature or prepared synthetically by
recombinant DNA procedures or other biological procedures. Suitable
probes are molecules which bind to the proteins or polypeptides of
the present invention. Such probes can be e.g., proteins, peptides,
lectins, or nucleic acid probes.
[0086] The vaccines and passive immunization agents of this
invention can be administered orally, parenterally, for example,
subcutaneously, intravenously, intramuscularly, intraperitoneally,
by intranasal instillation, or by application to mucous membranes,
such as, that of the nose, throat, and bronchial tubes. They may be
administered alone or with suitable pharmaceutical carriers, and
can be in solid or liquid form such as, tablets, capsules, powders,
solutions, suspensions, or emulsions.
[0087] The solid unit dosage forms can be of the conventional type.
The solid form can be a capsule, such as an ordinary gelatin type
containing the peptides, proteins, or polypeptides of the present
invention or the antibodies or binding portions thereof of the
present invention and a carrier, for example, lubricants and inert
fillers such as, lactose, sucrose, or cornstarch. In another
embodiment, these compounds are tableted with conventional tablet
bases such as lactose, sucrose, or cornstarch in combination with
binders like acacia, cornstarch, or gelatin, disintegrating agents
such as, cornstarch, potato starch, or alginic acid, and a
lubricant like stearic acid or magnesium stearate.
[0088] The peptides, proteins, or polypeptides of the present
invention or the antibodies or binding portions thereof or probes
of this invention may also be administered in injectable dosages by
solution or suspension of these materials in a physiologically
acceptable diluent with a pharmaceutical carrier. Such carriers
include sterile liquids such as water and oils, with or without the
addition of a surfactant and other pharmaceutically acceptable
adjuvants. Illustrative oils are those of petroleum, animal,
vegetable, or synthetic origin, for example, peanut oil, soybean
oil, or mineral oil. In general, water, saline, aqueous dextrose
and related sugar solution, and glycols such as, propylene glycol
or polyethylene glycol, are preferred liquid carriers, particularly
for injectable solutions.
[0089] For use as aerosols, the peptides, proteins, or polypeptides
of the present invention or the antibodies or binding portions
thereof or probes of the present invention in solution or
suspension may be packaged in a pressurized aerosol container
together with suitable propellants, for example, hydrocarbon
propellants like propane, butane, or isobutane with conventional
adjuvants. The materials of the present invention also may be
administered in a non-pressurized form such as in a nebulizer or
atomizer.
[0090] In yet another aspect of the present invention, the
peptides, proteins, or polypeptides of the present invention can be
used as antigens in diagnostic assays for the detection of
Mycobacterium tuberculosis body fluids. Alternatively, the
detection of that bacillus can be achieved with a diagnostic assay
employing antibodies or binding portions or probes thereof raised
by such antigens. Such techniques permit detection of Mycobacterium
tuberculosis in a sample of the following tissue or body fluids:
blood, spinal fluid, sputum, pleural fluids, urine, bronchial
alveolor lavage, lymph nodes, bone marrow, or other biopsied
materials.
[0091] In one embodiment, the assay system has a sandwich or
competitive format. Examples of suitable assays include an
enzyme-linked immunosorbent assay, a radioimmunoassay, a gel
diffusion precipitan reaction assay, an immunodiffusion assay, an
agglutination assay, a fluorescent immunoassay, a protein A
immunoassay, or an immunoelectrophoresis assay.
[0092] In an alternative diagnostic embodiment of the present
invention, the nucleotide sequences of the isolated DNA molecules
of the present invention may be used as a probe in nucleic acid
hybridization assays for the detection of Mycobacterium
tuberculosis in various patient body fluids. The nucleotide
sequences of the present invention may be used in any nucleic acid
hybridization assay system known in the art, including, but not
limited to, Southern blots (Southern, J. Mol. Biol., 98:508
(1975)); Northern blots (Thomas et al., Proc. Nat'l Acad. Sci. USA,
77:5201-05 (1980)); Colony blots (Grunstein et al., Proc. Nat'l
Acad. Sci. USA, 72:3961-65 (1975), which are hereby incorporated by
reference). Alternatively, the isolated DNA molecules of the
present invention can be used in a gene amplification detection
procedure (e.g., a polymerase chain reaction) . See H. A. Erlich
et. al., "Recent Advances in the Polymerase Chain Reaction",
Science 252:1643-51 (1991), which is hereby incorporated by
reference.
[0093] More generally, the molecular basis for the uptake
phenomenon achieved by Mycobacterium tuberculosis can be utilized
to effect uptake of other materials into mammalian cells. This is
achieved by utilizing the peptides, proteins, or polypeptides of
the present invention which effect cellular uptake (i.e. those
peptides, proteins, or polypeptides corresponding to the amino
acids having SEQ. ID. Nos. 2, 4, 8, and 10) in association with
such materials for uptake by mammalian cells. This phenomenon can
be used to introduce a wide variety of materials into such cells,
including antibiotics, DNA fragments, anti-neoplastic agents, and
mixtures thereof.
[0094] The opportunity for direct cell entry of antibiotics
constitutes a substantial advance, because they will be able to
kill intracellular Mycobacterium tuberculosis or other
intracellular pathogens (i.e. viruses, parasites, and fungal). One
approach for achieving such uptake is by impregnating microspheres
with antibiotics and then coating the spheres with the cellular
uptake peptides, proteins, or polypeptides of the present invention
in order to achieve such uptake. The microspheres can be
constructed from biodegradable biopolymers which effect sustained
release of the impregnated therapeutic. Such a system would be
particularly effective in delivering antituberculosis drugs by
aerosolization into the lungs where tubercule bacilli reside. For
example, drugs having in vitro utility against tuberculosis but
which are not used due to poor availability to mammalian cells
(e.g., amoxicillin-clavulanic acid) can be encapsulated in a
biopolymer coated with the protein, polypeptide, or peptide of the
present invention for aerosol delivery into the lungs of
tuberculosis patients. Alternatively, instead of utilizing
microspheres to transport antibiotics, such therapeutics can be
chemically linked to the cellular uptake peptides, proteins, or
polypeptides of the present invention.
[0095] This technology can be used to treat a wide array of
diseases caused by intracellular pathogens. For treatment of
tuberculosis, a repertoire of antibiotics, having themselves poor
cellular penetration but high activity against extracellular
Mycobacterium tuberculosis when tested in vitro, can be utilized in
conjunction with the cellular uptake proteins or polypeptides of
the present invention. In cancer treatment, intracellular delivery
of anti-neoplastic agents can be greatly enhanced by conjugating
such agents to the cellular uptake peptides, proteins, or
polypeptides of the present invention. This will enable reductions
in dosages for such agents and in their resulting toxicity.
[0096] Another aspect of the present invention is to utilize the
cellular uptake peptides, proteins, or polypeptides of the present
invention in gene therapy or in a genetic vaccine where pieces of
therapeutically or prophylactically useful DNA are conjugated at
their thymine residues to these peptides, proteins, or polypeptides
of the present invention via linker arms. As a result, genetic
material can be introduced into cells to correct genetic defects or
to produce a desired characteristic or products that serve as
immunogens.
EXAMPLES
Example 1
[0097] Preparation of and Screening for HeLa Cell Invasion
Clones
[0098] To identify the Mycobacterium tuberculosis DNA sequence that
encode mammalian cell entry, recombinant invasive clones were
constructed as follows: Mycobacterium tuberculosis H37Ra strain
(ATCC 25177) genome was digested with restriction enzymes Sau3 Al
and Eco Ri, and the DNA fragments were ligated into the Bam H1-Eco
R1 restriction sites of a phagemid vector pBluescript II
(Stratagene, La Jolla, Calif.). The recombinant vectors were
introduced into E. coli ELI-Blue (Stratagene) by electroporation.
We screened the recombinant strains for HeLa cell-invasive clones
by a method similar to that described by R. R. Isberg and S.
Falkow, Nature 317, 262 (1987), which is hereby incorporated by
reference.
[0099] One E. coli transformant XL1-Blue(pZX7), which harbored a
plasmid (pZX7) containing a 1535-base insert in the Bam HI-Eco RI
restriction enzyme sites of the pBluescript vector, was found by
the screening procedure to associate consistently with HeLa cells.
It was confirmed by transmission electron microscopy that this
clone entered HeLa cells (FIG. 1). FIG. 1A shows HeLa cells
infected with Mycobacterium tuberculosis strain H37Ra (ATCC 25177),
while the invasive recombinant strain E. coli XL1-Blue(pZX7) is
shown in FIGS. 1B and 1C. The cells were incubated with
Mycobacterium tuberculosis strain for 72 hours in FIG. 1A and with
XL1-Blue(pZX7) for 7.5 hours in FIG. 1B and FIG. 1C.
Internalization of this clone by HeLa cells was time-dependent
(FIG. 1B), with intracellular organisms visible as early as 3.5
hours after infection. Some phagosomes contained multiple organisms
(FIG. 1C), which suggested that the bacteria proliferated
intracellularly. Some of the internalized bacilli were surrounded
by a distinct ETZ, similar in appearance to the clear zone
surrounding Mycobacterium tuberculosis inside HeLa cells (FIG. 1A,
arrow). Whether this zone represents the ETZ often seen around
other pathogenic intracellular mycobacterial organisms (See P.
Draper and R. J. W. Rees, Nature 228, 860 (1970); N. Rastogi, Res.
Microbiol. 141, 217 (1990); T. Yamamoto, M. Nishimura, N. Harada,
T. Imaeda, Int. J. Lepr. 26, 111 (1958), which are hereby
incorporated by reference) or is an artifact of the preparation is
not clear.
[0100] Nonpathogenic E. coli XL1-Blue strains containing the vector
pBluescript or another pBluescript-derived recombinant vector
(pZN7) showed no association with HeLa cells after 7.5 hours.
[0101] To demonstrate that the invasive phenotype was indeed
encoded by the cloned Mycobacterium tuberculosis DNA fragment, we
transformed other nonpathogenic E. coli strains, specifically,
HB101, DH5u, and NM522, with pZX7. The constructs HB1O1(pZX7),
DH5u(pZX7), and NM522(pZX7) were invasive for HeLa cells. A
spontaneous loss of pZX7 on prolonged storage of XL1-Blue(pZX7) was
associated with loss of the invasive phenotype.
[0102] Four exonuclease III unidirectional deletion subclones of
pZX7 and the subclones Bam HI-Pst I (pZX7.1), Pst-I-HinD III
(pZX7.2), and Bam HI-Eco RI ([Zx7.7) was utilized for HeLa cell
association. The unidirectional deletion subclones of pZX7 were
generated using exonuclease III according to the manufacturer's
instruction (Erase-a-Base System, Promega, Madison, Wis.). The
plasmid pZX7 was double-digested with HinD III and Kpn I
restriction enzymes downstream from the Eco RI site of the Ban
HI-Eco RI DNA insert to generate a 5' protruding end adjacent to
the insert and a four-base 3' protruding end adjacent to the insert
and a four-base 3' protrusion at the opposite strand to protect it
from Exo III digestion. The digested plasmid was mixed with 300 U
of Exo III at 37.degree. C., and every 30 s 2.5.mu.l aliquots of
the Exo III digestion were transferred to tubes containing Si
nuclease to remove the remaining single-stranded tails. The S1
nuclease was inactivated by neutralization and heating at
70.degree. C. for 10 min. Klenow DNA polymerase was added to create
blunt ends which were ligated to circularize the
deletion-containing vectors. The ligation mixture was then used to
transform the competent E. coli XL1-Blue strain by electroporation.
These transformed strains were incubated for 6 hours with a HeLa
cell monolayer.
[0103] The results of this procedure are shown in FIG. 2. The black
bars represent the Mycobacterium tuberculosis DNA sequences, and
the white bars represent pBluescript sequences. As shown, the
strains of E. coli XL1-Blue harboring pZX7.3, pZX7.4, or pZX7.5
associated with HeLa cells in a pattern similar to that for E. coli
ZL1-Blue(pZX7), whereas the other subclones did not.
Example 2
[0104] Infection of Human Macrophages
[0105] Macrophage monolayers infected with the E. coli recombinant
clones of Example 1 were established on glass cover slips at the
bottom of polystyrene wells. They were initially infected with
.about.10 over-night-growth bacteria per macrophage cell for 1 or 2
hours followed by washing with phosphate-buffered saline (pH 7.4)
and incubation for an additional 1, 6, or 22 hours. Cultures were
performed at 37.degree. C. in RPMI-1640 medium (Gibco) with 2% AB
heat-inactivated human serum containing gentamicin (10 .mu.g/ml).
The gentamicin was included to kill the extracellular bacteria. The
macrophage monolayer was washed again and then lysed with sterile,
distilled water. The lysate was plated on tryptic soy agar medium
to obtain colony counts. For microscopy, the macrophage monolayer
was fixed with 100% methanol, stained with 10% Giemsa stain, and
examined by light microscopy or processed for electron
microscopy.
[0106] The monolayer that was infected for 1 hour only was examined
by light microscopy immediately after it was washed, fixed, and
stained. The macrophage lysate culture and light microscopy results
are shown in Table 1, infra. The percentage of infected macrophages
was calculated from counts of infected macrophages per 100 to 200
macrophage cells on a cover slip monolayer. Each E. coli strain was
tested four to six times for each time point, and the means of the
percentages of the cells infected by the E. coli recombinant clone
and the control strains XL1-Blue(pBluescript) and XL1-Blue(pZX7.3)
were compared by students T test.
[0107] FIG. 3 shows thin-section electron micrographs of human
macrophages exposed to the invasive recombinant E. coli clone
XL1-Blue(pZX7) for 3 hours (FIG. 3A) and 24 hours (FIG. 3B). In
FIG. 3C, the thin-section micrograph is of human macrophages
exposed to nonpathogenic E. coli XL1-Blue(pBluescript) for 24
hours. After 24 hours, bacilli were more numerous inside the cells,
compartmentalized, surrounded by multiple layers of a membrane
presumably of host origin (FIG. 3B). No bacteria could be seen
inside macrophages infected with E. coli (pBluescript) after 24
hours (FIG. 3C).
[0108] Table 1 shows the results obtained from this light
microscopy and culture study of human macrophage monolayer cells
infected with the HeLa cell-invasive E. coli XL1-Blue (pZX7),
subclone XL1-Blue (pZX7.3), and noninvasive XL1-Blue (p.
Bluescript). The colony-forming units (CFU) were determined per
milliliter of cell culture lysate. As shown, after 1 hour of
infection, the percentage of cells infected by the recombinant
clone (82.+-.8%) was more than five times that of cells infected by
XL1-Blue(pBluescript) (15.+-.6%, P<0.001).
11TABLE 1 lysate Ex- Percentage of infected cells CFU per
milliliters of posure (mean .+-. SEM) Culture (mean + SEM) (hours)
pBluescript pZX7.3 pZX7 pBluescript pZX7 1 15 .+-. 6 59 .+-. 10**
82 .+-. 8**** ND***** ND 3 9 .+-. 4 ND 55 .+-. 17 1800 .+-. 500
3500 .+-. 1700 8 4 .+-. 2 ND 35 .+-. 5 10 .+-. 5 1600 .+-. 400 24
12 .+-. 10 23 .+-. 8* 60 .+-. 13*** 3 .+-. 1 1300 .+-. 200 *p >
0.05, compared with pBluescript clone. 0.001, compared with
pBluescript or pZX7.3 clones. 0.05 compared # with pZX7.3 clone.
**p < 0.001, compared with pBluescript clone. ***p < 0.001,
compared with pBluescript or pZX7.3 clones. ****p < 0.0001,
compared with pBluescript clone, P < 0.05 compared with pZX7.3
clone. *****ND means not determined.
[0109] This observation suggests that the cloned Mycobacterium
tuberculosis DNA sequences facilitate bacterial uptake at
quantities above the background phagocytic activity of the
macrophage cells. After 24 hours of infection, 12% (.+-.10%) of the
macrophages exposed to XL1-Blue(pBluescript) and 60% (.+-.13%) of
the cells exposed to XL1-Blue(pZX7) were infected (P<0.001). As
demonstrated in Table 1, culture of the lysate of macrophages that
had been infected for 24 hours showed that the intracellular E.
coli XL1-Blue(pZX7) strains were viable.
[0110] In comparing capacity of XL1-Blue(pZX7),
XL1-Blue(pBluescript), and one HeLa cell-invasive deletional
derivative, E. coli XL1-Blue(pZX7.3), to infect macrophages from
Table 1, at 1 hour of infection, the invasive capacity of E. coli
XL1-Blue(pZX7.3) was four times that of XL1-Blue(pBluescript)
(P<0.001), but by 24 hours the difference was no longer
apparent. Thus, the DNA sequences associated with HeLa cell
invasion are responsible for increased uptake by the macrophage,
and the sequences that confer survival within the macrophage are
located downstream of those necessary for mammalian cell entry.
Example 3
[0111] Homology Analysis
[0112] The Bam Hi-Eco Ri DNA fragment was sequenced by the chain
termination-method, described in F. Sanger, et. al., "DNA
Sequencing with Chain-Terminating Inhibitors," Proc. Nat. Acad.
Sci., 74:5463-67, which is hereby incorporated by reference, and
found to have 1535 base pairs [European Molecular Biology
Laboratory (EMBL) accession number X70901]. The sequence showed no
homology with any of the DNA sequences in the database of GenBank
(R72.0) or EMBL (R31.0). No obvious procaryotic promoter consensus
sequence could be discerned. If we assume that Mycobacterium
tuberculosis uses the common prokaryotic termination codon
sequences, amino acid sequence homologies can be identified. A
region near the NH.sub.2-terminus of the deduced sequence of one
potential open reading frame was found to share (i) 27% identity
with an 80-residue NH.sub.2-terminus region of internalin, a
protein encoded by Listeria monocytogenes that is associated with
mammalian cell entry (A. B. Hartman, M. Venkatesan, E. V. Oaks, J.
M. Buysse, J. Bacteriol, 172, 1905 (1990), which is hereby
incorporated by reference); (ii) 20% identity with a 145-residue
region of the IpaH gene product of the invasiveness plasmid of
Shigella (B. E. Anderson, G. A. McDonald, D. C. Jones, R. L.
Regnery, Infect. Immun. 58, 2760 (1990), which is hereby
incorporated by reference); and (iii) 18% identity with a
176-residue region of human .beta.-adaptin, a plasma membrane
protein that links clathrin to receptors in coated vesicles which
are responsible for receptor-mediated endocytosis (S. Ponnambalam,
M.S. Robinson, A. P. Jackson, L. Peiper, P. Parham, J. Biol. Chem.
265, 4814 (1990) and J. L. Goldstein, M. S. Brown, R. G. W.
Anderson, D. W. Russell, W. J. Schneider, Annu. Rev. Cell Biol. 1,1
(1985), which are hereby incorporated by reference). When aligned
against the invasin protein of Yersinia pseudotuberculosis, the
region associated with cell entry was 19% identical with a
100-residue region near the invasion COOH-terminus (R. R. Isberg,
D. L. Voorhis, S. Falkow, Cell 50, 769 (1987), which is hereby
incorporated by reference). The functional significance of these
alignments is not clear.
Example 4
[0113] Functional Analysis of 52kD Polypeptide
[0114] Protein fractions analyzed by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) were prepared as follows: A 5-ml aliquot
of bacterial overnight growth (adjusted to absorbance at 550 nm at
optical density 600) in tryptic soy broth containing ampicillin
(100 .mu.g/ml) was harvested by centrifugation. We then sonicated
the bacterial pellet in 1.5 ml of 10 mM tris-HCI buffer (pH 8.0)
containing 5 mM MgCI.sub.2. The sonicate was centrifuged for 25 min
at 12,000 rpm in a microcentrifuge (Eppendorf model 5415C) at
4.degree. C. Acetone was added to 600 .mu.l of the supernatant in a
fresh microcentrifuge tube (60% v/v), and the mixture was
centrifuged for 25 min. at 14,000 rpm at 4.degree. C. The pellet
was resuspended in 20 .mu.l of distilled water and 20 .mu.l of
Laemmli's boiling buffer, heated over boiling water for 5 min. and
analyzed by SDS-PAGE. The bacterial debris containing the outer
membrane fraction after the first centrifugation was resuspended in
100 .mu.l of water and 100 .mu.l of 15 mM tris-HCI buffer (pH 8.0)
containing 7.5 mM MgCI.sub.2 and 3% (v/v) Triton X-100 and
centrifuged for 25 min. at 14,000 rpm. The pellet was resuspended
in 25 .mu.l of water and 25 .mu.l of boiling buffer and boiled and
analyzed a 20-.mu.l aliquot of the sample by SDS-PAGE.
[0115] The SDS-PAGE (i.e., SDS-polyacrylamide gel electrophoresis)
of acetone precipitated a soluble fraction of bacterial cell
sonicate. The polypeptides were analyzed in a 9% gel (left):
molecular size standards (lane 1), E. coli XL1-BBlue with a vector
(pZN) containing an unrelated Mycobacterium tuberculosis DNA
fragment between the Bam HI-Eco RI pEluescript cloning sites (lane
2), and XLl-Blue(pZX7) (land 3). Analysis in an 8% gel (right):
XL1-Blue containing a vector (pZX7.8) with a two base frameshift
introduced 12 bases upstream from the Bam HI cloning site in pZX7
(lane 1) and XL1-Blue(PZX7) (lane 2). Molecular sizes are indicated
at the far right. We detected a 52-kD polypeptide in the soluble
protein fraction of XLl-Blue(pZX7) (arrow). A protein of about 50
kD is expressed by XLl-Blue containing pZX7.8. The expression of
the 52-kD protein was always associated with HeLa cell interaction
of the recombinant E. coli clone.
[0116] From the SDS-PAGE results of FIG. 4, it can be concluded
that a soluble fraction of the bacterial cell sonicate of
XLl-Blue(pZX7) contained a 52-kD polypeptide that was not detected
in the soluble fraction of XLl-Blue with a pEluescript-derived
vector (pZN7) harboring an unrelated Mycobacterium tuberculosis DNA
fragment. A two-base frameshift, introduced by blunt-end ligation
after the 5' protruding end had been filled with Klenow DNA
polymerase at the Xba I site 12 bases upstream from the Bam HI
cloning site in pZX7 (confirmed by sequencing), led to loss of
association with HeLa cells of the E. coli XL1-Blue containing this
plasmid (pZX7.8). This clone did not express the 52-kD protein, but
a new polypeptide of lower molecular mass was detected in the
soluble fraction. A spontaneous loss of the capacity to associate
with HeLa cells after prolonged storage of XL1-Blue(pZX7) was
accompanied by loss of the 52-kD protein. Hence, this 52-kD protein
is likely to be a product expressed by the cloned Mycobacterium
tuberculosis DNA fragment. There were no detectable differences in
the bacterial outer membrane polypeptide fractions.
Example 5
[0117] Subcloning The Open Reading Frame (ORF-1) That Encodes A
Protein That Mediates Entry Of Mycobacterium Tuberculosis Into
Mammalian Cells
[0118] The nucleotide sequence corresponding to SEQ. ID. No. 3
(i.e. ORF-1) was subcloned into the EcoRI and HinDIII endonuclease
sites of pET vectors (pET23a, b, c, from Novagen). This was done by
subcloning a PCR-amplified product of the ORF-1 fragment. The
primers used to amplify the ORF-1 are as follows: EcoRI-primer:
5'-GGGGAATTCA TGTGAACGCC GACATCAA (SEQ. ID. No. 11);
HinDIII-primer: 5'-GGGAAGCTTA TTGCGGCAGC CCCGGCGTC (SEQ. ID. No.
12). Extracted DNA from M. tuberculosis strain H37Ra (ATCC 25177)
was amplified for 30 cycles using the following PCR conditions:
denaturation at 94.degree. C. for 1 min, primer annealing at
56.degree. C. for 2 min, and primer extension at 72.degree. C. for
1 min. The amplified DNA was resolved by electrophoresis in 1.8%
agarose gel, and, after visualization under UV illumination, the
amplified DNA was removed from the gel using QIAEX, according to
the manufacturer's instructions. The DNA was then digested with
EcoRI and HinDIII in the same digestion buffer.
[0119] The pET vectors were also digested with EcoRI and HinDIII
endonucleases, resolved in 1% agarose, and the linearized vector
was removed from the gel, and mixed with the EcoRI/HinDIII digest
of the PCR-amplified ORF-1 DNA fragment for a ligation
reaction.
[0120] The ligation reaction was performed as follows: To a mixture
containing 5 .mu.l of the digested PCR-amplified DNA product and 3
.mu.l of the vector DNA digest, 1 .mu.l of 10X T4 ligase buffer
(New England BioLabs) and 1 .mu.l of T4 ligase (15 U) were added.
The mixture was incubated at room temperature for 4 hrs. A 1.5.mu.l
aliquot of the ligation mixture was electroporated into E. coli
strain BL21(DE3), and the E. coli was inoculated onto
ampicillin-containing (200 .mu.g/ml) agar plates for incubation
overnight at 37.degree. C. Representative colonies from each of the
pET23 constructs (pET23a-ORF1, pET23b-ORF1, pET23c-ORF1) were
tested for their association with HeLa cells as described
elsewhere. The strains were tested with and without induction by
IPTG.
Example 6
[0121] SDS-Polyacrylamide Gel Electrophoresis Analysis Of The
Protein Expressed By ORF-1
[0122] To express the protein encoded by ORF-1, the pET23
recombinant BL21(DE3) E. coli strains were first grown overnight in
5 ml of ampicillin containing tryptic soy broth (TSB) medium. The
following day, a 500-.mu.l sample was pelleted and resuspended in 5
ml of TSB containing ampicillin (200 .mu.g/ml), and incubated for 3
hrs. Then, 50 .mu.l of IPTG (40 mM) was added to the growth and
incubated for additional 2 hrs at 37C. A 1-ml bacterial suspension
(OD=500 at Abs.sub.600) was pelleted, and the pellet was
resuspended in 50 .mu.l water and 50 .mu.l of Laemmli's boiling
buffer and boiled for 5 min. A 15 .mu.l-aliquot of the boiled
sample was loaded onto 12% SDS-polyacrylamide gel, and resolved
electrophoretically. BL21(DE3) containing a pET vector was treated
similarly as a control in these experiments.
[0123] The SDS-PAGE revealed a protein at position around 23-28 KDa
expressed by BL21(DE3)(pET23c-ORF1), that was not expressed by any
of the other pET23 constructs or the control BL21(DE3)(pET23c)
strain. Even without induction by IPTG, some expression of the
protein was evident (FIG. 5). The same recombinant strain BL21(DE3)
(pET23c-ORF1) showed a strong association with HeLa cells also.
Hence, the expressed product of ORF-1 has been shown to be
sufficient to confer HeLa cell association.
Example 7
[0124] N-terminal Analysis Of The Recombinant ORF-1 Protein
[0125] The IPTG-treated BL21(DE3) (pET23c-ORFl) strain was prepared
as described above for SDS-PAGE. Eight lanes were loaded with the
same bacterial lysate, and one lane was loaded with the control E.
coli lysate. After electrophoresis, the resolved proteins were
transferred onto a piece of PVDF membrane (Immobilon, Millipore),
using an electro-blotting apparatus (IDEA Scientific Company). The
membrane was stained with Coomassie Blue for 2 min and destained
until the transferred protein bands became visible. A protein
fraction of 25-28 KDa in the recombinant E. coli lanes, not present
in the control E. coli lane, was cut out, and sent to Stanford
University Protein and Nucleotide Facility for microsequencing of
the N-terminus. The N-terminus contained the pET vector's T7 tag
amino acid sequence (position 1 to 15), followed by Val, Asn, Ala,
Asp, Ile, which confirms the N-terminus amino acid sequence deduced
from the nucleotide sequence of ORF-1.
Example 8
[0126] Coating Of Latex Beads With The Recombinant Protein To Study
HeLa Cell Association Of The Beads
[0127] A crude preparation of the 23-28 kDa protein encoded by
ORF-1 was obtained from BL21(DE3)(pET23c-ORFl) as follows: The
protein was expressed as described above by IPTG induction. After
induction, the bacterial suspension was mixed to a final
concentration of 10% (vol/vol) in a Tris buffer (pH 8.0) containing
100 mM NaCl and 1 mM EDTA. Lysozyme was added to the solution to a
final concentration of 1 mg/ml, and the cells were incubated at
room temperature for 20 min. The cells were then centrifuged at
5000 g for 10 min, and the supernatant was discarded. The pellet
was transferred to ice, and resuspended in 5 ml of ice-cold 50 mM
Tris buffer (pH 8.0) containing 100 mM NaCl, 1 mM EDTA, and 0.1%
sodium deoxycholate. MgCl.sub.2 and DNAseI were added to final
concentrations of 8 mM and 10 .mu.g/ml, respectively. Incubation
was carried out on ice until the viscocity disappeared. The
inclusion body constituting the material in the suspension was
removed by centrifugation at 10,000 g for 10 min. The resulting
pellet was washed by resuspending in 5 ml of 50 mM Tris buffer
containing 1 NP-40, 100 mM NaCl, and 1 mM EDTA, followed by washing
in the same buffer not containing NP-40. An aliquot of the pellet
material was examined by SDS-PAGE for the presence of the
recombinant protein.
[0128] The remainder of the pellet was dissolved in 2 ml of 6 M
guanidium-HCL (GuHCl) in a 25 mM HEPES buffer (pH 7.6) containing
100 mM KCl, 0.1 mM EDTA, 125 mM MgCl.sub.2, 10% glycerol, and 0.1%
NP-40 (HEMGN buffer), that contained protease inhibitors (1 mM DTT,
2 .mu.g/ml aprotinin, 1 .mu.g/ml leupeptin, 1 .mu.g/ml pepstatin,
0.1 mM PMSF, and 0.1 mM Na-metabisulfite). The solubilized protein
was subjected to sequential dialysis against the HEMGN buffer
lacking 6 M GuHCl at 4C over a period of 2 days. For control, the
same procedure was carried out with the cells of E. coli
BL21(DE3)(pET23c). The protein concentration was determined by the
BCA protocol.
[0129] A 2-.mu.l sample of 10% aqueous suspension of 0.3 .mu.m
polystyrene latex beads (Sigma) was added to 1 ml of 100 .mu.g/ml
protein solution in PBS (pH 7.5). The beads were incubated with the
protein solution overnight at 37C with constant shaking, and
subjected to periodic, brief sonication to disperse the clumps. A
100-.mu.l suspension of the beads was then added to HeLa cell
monolayers grown in MEM (containing 10% fetal calf serum) on round
glass coverslips in 24-well tissue culture plates. The controls
included beads incubated in PBS alone, in PBS containing 1% BSA,
and beads coated with the protein preparation from the control E.
coli strain described above. The HeLa cell monolayers in 2 ml of
MEM per well were incubated for 5 hrs at 37C, then washed 5 times
with PBS, and fixed with 100% methanol for 30 min. The cells were
then stained with 10% Giemsa for 20 min and examined by light
microscope.
[0130] HeLa cells were also prepared for examination by
transmission electron microscopy. The HeLa cell monolayers after
the 5-hr incubation period were fixed in 2% glutealdehyde in PBS
(pH 7.5) for 3 hrs, then scraped off, and resuspended in the same
glutealdehyde buffer. The cells were then gently pelleted, and the
pellet was prepared for sectioning by a standard protocol for
transmission electron microscopy. One result is shown in FIG.
6.
Example 9 - Raising A Polyclonal Antisera To The Recombinant
Protein
[0131] A lysate of E. coli BL21(DE3) (pET23c-ORF1) expressing the
23-28 kDa protein was resolved by 12% SDS-PAGE in multiple wells,
and the protein was excised from the gel. The pieces of acrylamide
gel containing the protein was then pulverized using a mortar and
pestle, and resuspended in 2 ml of sterile PBS (pH 7.5). A rough
estimate of the protein concentration was made by the BCA method.
Six-month-old NZW female rabbits were injected subcutaneously at
7-8 sites with approximately 20 .mu.g of the antigen suspension per
site. The rabbits were boosted with the same amount of antigen
after 4 weeks and 6 weeks of the first injection. Serum was
collected from blood obtained after 2 weeks of the last booster
injection. Its reactivity to the recombinant protein was examined
by Western blotting. The immune antiserum diluted 1:10,000 was able
to detect less than 1 .mu.g of the protein bound to nitrocellulose
membrane. The antibody recognized the 23-28 kilodalton polypeptide
of Example 7 expressed by E. coli BL21 (DE3) (pET23c-ORF1) and a 45
kilodalton protein in a cell lysate of Mycobacterium tuberculosis
strain H37Ra. The reason that the 23-28 kilodalton protein
expressed in E. coli is smaller than the 45 kilodalton protein in
the cell lysate of the native organism is believed to be one of the
following: (1) the recombinant protein is truncated or (2) the
native protein is post-translationally modified.
Example 10
[0132] Analysis For The Presence of IS6110
[0133] A partial digest of the genomic DNA of Mycobacterium
tuberculosis strain H37Ra (ATCC 25177) was prepared with Sau3AI and
EcoRI restriction enzymes. Because the H37Ra strain contains
multiple copies of IS6110, described by U.S. Pat. No. 5,183,737 to
Crawford, et al., and IS6110 does not have an EcoRI site, the
digest would contain several DNA fragments containing IS6110. The
DNA fragments were ligated into the BamHI-EcoRI restriction sites
of the vector pBluescript II to create a recombinant library. The
recombinant vectors were then electroporated into E. coli XL1-Blue.
These recombinant E. coli strains were then screened for invasive
clones by the method described elsewhere in this application.
[0134] After the initial screening using HeLa cells, 15 E. coli
colonies were recovered. Only one of these consistently showed
association with HeLa cells. This is the previously described
strain XL1-Blue (pZX7). Others showed either weak or no association
with HeLa cells when tested multiple times. These other strains
were recently tested for the presence of IS6110 by a probe
generated from PCR-amplification of a 245-bp region within IS6110
using the following primers: INS1:5'-CGTGAGGGCATCGAGGTGGC (SEQ. ID.
No. 13) and INS2:51-GCGTAGGCGTCGGTGACAAA (SEQ. ID. No. 14).
[0135] None contained the IS6110 sequences. Furthermore, the
absence of consistent and strong association of other clones with
HeLa cells suggests that the sequence contained within pZX7 is the
only sequence among the DNA fragments in this genomic library that
encodes mammalian cell entry.
Example 11
[0136] Identification of Active Domains of the 23-28 KDa Protein
Encoded by ORF-1
[0137] Overlapping peptides of 20-22 amino acids spanning the
entire 23-28 KDa protein encoded by ORF-1 ("Mcep") were
constructed. One peptide comprised of 60 amino acids at the
N-terminus was also constructed. Peptides were synthesized using an
ABI 430A peptide synthesizer and optimized t-Boc chemistry as
described by the manufacturer, then cleaved from the resin by
hydrof luoric acid (HF). The peptides were purified by
reversed-phase high performance liquid chromatography (RP-HPLC) on
a Vydac C4 semi-preparative column (1.times.30 cm) using a 10 to
50% acetonitrile gradient in 0.1% trifluoryl acetic acid (TFA)
developed over 40 minutes at a flow rate of 2 mL/min. All synthetic
peptides were >95% pure as judged by analytical HPLC. Amino acid
composition analyses of these peptides performed on a Waters
Pico-Tag system were in good agreement with their theoretical
compositions. These peptides are listed below in Table 2.
12 TABLE 2 Position of residues on Peptides Mcep (invasin) Inv1
1-21 Inv2 13-34 Inv3 25-46 Inv4 38-60 Inv5 1-60 Inv6 56-80 Inv7
76-109 Inv8 113-139 Inv9 134-166 Inv10 159-188 Inv11 184-209
[0138] Each peptide was used to coat latex beads of varying
diameters. Two of the peptides promoted entry of the beads into
HeLa cells as efficiently as the whole Mcep (FIG. 7). These were
Inv3 peptide (22 amino acids) (SEQ. ID. No. 10) and Inv5 peptide
(60 amino acids) (SEQ. ID. No. 8). The Inv3 sequence is contained
within the Inv5 peptide sequence. Internalization of the beads was
achieved at concentrations of the peptides as low as 25 nM.
Solubilized Inv3 or InvS peptides competitively blocked the uptake
of Mcep-coated beads (coated with 50 nmoles of Mcep), while
peptides based on sequences from other regions of the protein did
not.
[0139] The 22-amino acid sequence was found in the PIR database to
be 41% identical to a domain in the fusion protein of an obscure
paramyxovirus isolated from monkeys suffering from a respiratory
illness in Japan called Murayama virus or MrV. Kusagawa, et al.,
"Antigenic and Molecular Properties of Murayana Virus Isolated from
Cynomologous Monkeys: Virus is Closely Related to Avian
Paramyxovirus Type 2," Virolocy 194:828-32(1993), which is hereby
incorporated by reference. A 12-amino acid region upstream of the
Inv3 peptide was 50% identical to the corresponding upstream region
of the fusion domain (F1) of this virus.
Example 12
[0140] Evaluation of Inv3 Derivatives
[0141] In order to determine which amino acids in the Inv3 peptide
were critical for the cell entry function, additional peptides were
designed with alterations in what was thought to be important amino
acids. Evaluations were made by coating each of the peptide
derivatives on latex beads, incubating the beads with HeLa cells,
and evaluating whether uptake of the beads occurred, in accordance
with Examples 8 and 11. The following alterations were made:
13 Uptake Inv3 T K R R I T P K D V I D V R S V T T E I N T +
Derivatives Inv3.1: E - Inv3.2: E E - Inv3.3: D - Inv3.4: K +
Inv3.5: K + Inv3.6: -----------Omit-------.fw- darw. - Inv3.7: E -
Ihv3.8: A + Inv3.9: AA -
[0142] The conclusion that can be made from this study is that
there are critical amino acid residues at positions 27, 28, and 38
of the amino acid sequence corresponding to SEQ. ID. No. 4 which
are arginine. At the 27 and 28 positions, changing the positively
charged arginine residue to a negatively charged glutamine or a
neutrally charged alanine prevented delivery of microspheres into
HeLa cells. Similarly, it appears that the amino acid at position
26 is not critical, because conversion of the positively charged
lysine residue to negatively charged glutamine prevented uptake
while conversion to neutrally charged alanine permitted uptake.
Meanwhile, when the lysine residue at position 32 was converted to
a negatively charged aspartic acid, uptake was prevented. Changing
the negatively charged aspartic acid and glutamine residues at
positions 33 and 43 to positively charged lysine did not affect
uptake.
[0143] Although the invention has been described in detail for the
purpose of illustration, it is understood that such detail is
solely for that purpose, and variations can be made therein by
those skilled in the art without departing from the spirit and
scope of the invention which is defined by the following claims.
Sequence CWU 1
1
* * * * *