U.S. patent application number 10/226935 was filed with the patent office on 2002-12-26 for vaccine against mycobacterial infections.
Invention is credited to Lowrie, Douglas Bruce.
Application Number | 20020198168 10/226935 |
Document ID | / |
Family ID | 10755365 |
Filed Date | 2002-12-26 |
United States Patent
Application |
20020198168 |
Kind Code |
A1 |
Lowrie, Douglas Bruce |
December 26, 2002 |
Vaccine against Mycobacterial infections
Abstract
A naked nucleic acid construct comprising a coding sequence
which encodes a mycobacterial stress protein or proline-rich
antigen or an antigenically effective fragment thereof operably
linked to a promoter capable of expressing the said coding sequence
in a mammalian host cell is useful as a vaccine against a
mycobacterial infection such as tuberculosis and leprosy.
Inventors: |
Lowrie, Douglas Bruce;
(London, GB) |
Correspondence
Address: |
JOHN S. PRATT, ESQ
KILPATRICK STOCKTON, LLP
1100 PEACHTREE STREET
SUITE 2800
ATLANTA
GA
30309
US
|
Family ID: |
10755365 |
Appl. No.: |
10/226935 |
Filed: |
August 23, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10226935 |
Aug 23, 2002 |
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08737487 |
Nov 15, 1996 |
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08737487 |
Nov 15, 1996 |
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PCT/GB95/01119 |
May 18, 1995 |
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Current U.S.
Class: |
514/44R ;
536/23.7 |
Current CPC
Class: |
A61P 31/04 20180101;
A61K 39/00 20130101; C07K 14/35 20130101; A61P 31/06 20180101; A61P
31/08 20180101; A61K 2039/51 20130101 |
Class at
Publication: |
514/44 ;
536/23.7 |
International
Class: |
C07H 021/04; A61K
048/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 18, 1994 |
GB |
9409985.0 |
Claims
1. Use of a naked nucleic acid construct comprising a coding
sequence which encodes a mycobacterial stress protein or
proline-rich antigen or an antigenically effective fragment thereof
operably linked to a promoter capable of expressing the said coding
sequence in a mammalian host cell, in the manufacture of a
medicament for use as a vaccine against a mycobacterial
infection.
2. Use according to claim 1 wherein the coding sequence of the
naked nucleic acid construct encodes a mycobacterial heat-shock
protein.
3. Use according to claim 2 wherein the coding sequence of the
naked nucleic acid construct encodes the 70 kDa, 65 kDa or 10 kDa
heat shock protein of Mycobacterium tuberculosis, Mycobacterium
leprae or Mycobacterium bovis or an antigenically effective
fragment thereof.
4. Use according to claim 3 wherein the coding sequence of the
naked nucleic acid construct encodes the Mycobacterium leprae 65
kDa protein.
5. Use according to claim 1 wherein the coding sequence of the
naked nucleic acid construct encodes a proline-rich antigen of
Mycobacterium tuberculosis, Mycobacterium leprae or Mycobacterium
bovis.
6. Use according to claim 5 wherein the coding sequence of the
naked nucleic acid construct encodes the 36 kDa proline-rich
antigen of Mycobacterium leprae.
7. Use according to any one of the preceding claims wherein the
naked nucleic acid construct is a DNA construct.
8. Use according to the any one of the preceding claims wherein the
naked nucleic acid construct is plasmid.
9. Use according to any one of claims 1 to 6 wherein the naked
nucleic acid construct is an RNA construct.
10. A naked nucleic acid construct as defined in any one of the
preceding claims for use as a vaccine against a mycobacterial
infection.
11. A vaccine composition comprising a naked nucleic acid construct
as defined in any one of claims 1 to 9 and an acceptable carrier or
diluent.
12. A method of vaccinating a mammalian host against a
mycobacterial infection, which method comprises administering to
the host an effective amount of a naked nucleic acid construct as
defined in any one of claims 1 to 8.
13. Bone marrow cells transfected with a nucleic acid construct
comprising a coding sequence which encodes a mycobacterial stress
protein or proline-rich antigen or an antigenically effective
fragment thereof operably linked to a promoter capable of
expressing the said coding sequence in bone marrow cells.
14. A method of vaccinating a mammalian host against a
mycobacterial infection, which method comprises administering to
the host an effective amount of bone marrow cells as defined in
claim 13.
15. A naked nucleic acid construct as defined in claim 1, or in any
one of claims 5 to 9 when dependent thereon, wherein the coding
sequence encodes a mycobacterial proline-rich antigen or an
antigenically effective fragment thereof.
Description
[0001] This invention relates to vaccines against mycobacterial
infections such as tuberculosis and leprosy.
[0002] Despite its central position in classical immunology,
surprisingly little is known of how a protective cell-mediated
immune response is either acquired or expressed against
tuberculosis or leprosy. It is not know why vaccination with live
bacille Calmette-Guerin (BCG) is highly protective in only some
human populations or why, in contrast to live BCG, injections of
dead BCG or antigenic components, even in large amounts and with
adjuvants, confer only slight protection in animals.
[0003] In an attempt to develop an alternative vaccine based on the
Mycobacterium leprae 65 kDa heat shock protein (MLhsp65) antigen
(Mehra et al (1986): Proc. Natl. Acad. Sci. USA; 83, 7014-7017), we
have now stably transfected bone marrow cells with an expression
vector encoding this antigen. When the transfected bone marrow
cells were injected into mice, the mice were found to be resistant
to infection by Mycobacterium tuberculosis, the causative agent of
tuberculosis. Further, we have injected mice with naked DNA
encoding MLhsp65 or the Mycobacterium leprae 36 kDa proline
rich-antigen (Thole et al, Infection and Immunity (1990) 58,
80-87). These mice were also found to be resistant to infection by
Mycobacterium tuberculosis.
[0004] These findings have general applicability. Accordingly, the
present invention provides use of a naked nucleic acid construct
comprising a coding sequence which encodes a mycobacterial stress
protein or proline-rich antigen or an antigenically effective
fragment thereof operably linked to a promoter capable of
expressing the said coding sequence in a mammalian host cell, in
the manufacture of a medicament for use as a vaccine against a
mycobacterial infection.
[0005] The invention also provides:
[0006] such a naked nucleic acid construct for use as a vaccine
against a mycobacterial infection;
[0007] a vaccine composition comprising such a naked nucleic acid
construct and an acceptable carrier or diluent;
[0008] a method of vaccinating a mammalian host against a
mycobacterial infection, which method comprises administering to
the host an effective amount of such a naked nucleic acid
construct;
[0009] bone marrow cells transfected with a nucleic acid construct
comprising a coding sequence which encodes a mycobacterial stress
protein or proline-rich antigen or an antigenically effective
fragment thereof operably linked to a promoter capable of
expressing the said coding sequence in bone marrow cells;
[0010] a method of vaccinating a mammalian host against a
mycobacterial infection, which method comprises administering to
the host an effective amount of such transfected bone marrow cells;
and
[0011] a naked nucleic acid construct as above wherein the coding
sequence encodes a mycobacterial proline-rich antigen or an
antigenically effective fragment thereof.
[0012] The naked nucleic acid construct comprises a coding sequence
which encodes a mycobacterial stress protein or a mycobacterial
proline rich-antigen or an antigenically effective fragment thereof
operably linked to a promoter capable of directing expression of
the said coding sequence in a mammalian host cell. Nucleic acid
encoding at least one further mycobacterial protein or fragment
thereof operably linked to a promoter may be included in the
construct. Typically, the thus encoded further mycobacterial
protein or fragment thereof will be an antigenic protein or an
antigenic fragment thereof. The further mycobacterial protein or
fragment thereof may be a further mycobacterial stress protein or
proline-rich antigen or antigenic fragment thereof.
[0013] The naked nucleic acid construct is typically cell-free and
virus-free. It is typically in isolated form. It may be purified.
Although it is preferred that a construct is DNA, it may also be
RNA or a modified nucleic acid. The nucleic acid may contain
modifications in its backbone and possibly additions at either the
5' or 3', or both, ends of the molecule in the case of linear, as
opposed to circular, constructs. This may assist in prolonging the
life of the nucleic acid when taken up by host cells, for example,
muscle cells which may enhance the potency of the construct. Known
modifications to nucleic acid molecules include the provision of
methylphosphonate and phosphorothioate backbones and addition of
acridine or polylysine chains at the 3' and/or 5' ends of the
molecule.
[0014] The mycobacterial stress protein encoded by the nucleic acid
constructs of the present invention is generally one whose
expression increases substantially when the mycobacterium from
which it is derived is placed under environmental stress.
Typically, the mycobacterial stress protein is a heat shock
protein, for example a protein whose expression increases
substantially when the bacterium from which it is derived is
subjected to a high temperature, for example 42.degree. C. or
greater.
[0015] The mycobacterial stress protein is typically derived from
Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium
bovis, Mycobacterium aviumor Mycobacterium vaccae. Suitable
proteins include the 70, 65 and 10 kDa heat shock proteins of
Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium
bovis, Mycobacterium avium or Mycobacterium vaccae. Of these, the
65 kDa heat shock proteins of Mycobacterium tuberculosis,
Mycobacterium leprae and Mycobacterium bovis are preferred, the
heat shock proteins of Mycobacterium leprae being particularly
preferred.
[0016] The mycobacterial proline-rich antigen may be a proline-rich
antigen of Mycobacterium tuberculosis, Mycobacterium leprae,
Mycobacterium bovis, Mycobacterium avium or Mycobacterium vaccae. A
suitable proline-rich antigen is the 36 kDa proline-rich antigen of
Mycobacterium leprae.
[0017] An antigenic fragment of a mycobacterial stress protein or
proline-rich antigen preferably contains a minimum of five, six,
seven, eight, nine, ten, fifteen, twenty, thirty, forty or fifty
amino acids. The fragment may be up to ten, twenty, thirty, forty
or fifty amino acids long. Alternatively, up to twenty or up to ten
amino acid residues may have been omitted from the amino- and/or
carboxy-terminus of the stress protein or proline-rich antigen.
[0018] The antigenic sites of the mycobacterial stress protein or
proline-rich antigen may be identified using standard procedures.
These may involve fragmentation of the polypeptide itself using
proteolytic enzymes or chemical agents and then determining the
ability of each fragment to bind to antibodies or to provoke an
immune response when inoculated into an animal or suitable in vitro
model system (Strohmaier et al, J. Gen. Virol., 1982, 59,
205-306).
[0019] Alternatively, the DNA encoding the mycobacterial stress
protein or proline-rich antigen may be fragmented by restriction
enzyme digestion or other well-known techniques and then introduced
into an expression system to produce fragments. These fragments may
be fused to a polypeptide usually a polypeptide of bacterial
origin. The resulting fragments are assessed as described
previously (Spence et al, J. Gen. Virol., 1989, 70, 2843-51; Smith
et al, Gene, 1984, 29, 263-9).
[0020] Another approach is to chemically synthesise short peptide
fragments (3-20 amino acids long; conventionally 6 amino acids
long) which cover the entire sequence of the full-length
polypeptide with each peptide overlapping the adjacent peptide.
This overlap can be from 1-10 amino acids but ideally is n-1 amino
acids where n is length of the peptide; Geysen et al, Proc. Natl.
Acad. Sci, 1984, 81, 3998-4002. Each peptide is then assessed as
described previously except that the peptide is usually first
coupled to some carrier molecule to facilitate the induction of an
immune response.
[0021] Finally, there are predictive methods which involve analysis
of the sequence for particular features, e.g. hydrophilicity,
thought to be associated with immunologically important sites (Hopp
and Woods, Proc. Natl. Acad. Sci., 1981, 78, 3824-8; Berzofsky,
Science, 1985, 229, 932-40). These predictions may then be tested
using the recombinant polypeptide or peptide approaches described
previously.
[0022] The nucleic acid sequence encoding the mycobacterial shock
protein or proline-rich antigen or fragment thereof is typically
included within a replicable expression vector. Such an expression
vector comprises an origin of replication so that the vector can be
replicated in a host cell such as a bacterial host cell, a promoter
for the expression of the nucleic acid sequence and optionally a
regulator of the promoter. The vector may contain one or more
selectable marker genes, for example an ampicillin resistance gene
for the identification of bacterial transformants or a neomycin
resistance gene for the identification of mammalian cell
transformants. Optionally, the nucleic acid construct may also
comprise an enhancer for the promoter. The construct may also
comprise a polyadenylation signal operably linked 3' to the nucleic
acid encoding the functional protein. The construct may also
comprise a terminator 3' to the sequence encoding the mycobacterial
stress protein or fragment thereof. The construct may also comprise
one or more introns or other coding sequences 3' to the sequence
encoding the mycobacterial stress protein or fragment thereof. The
intron or introns may be from the host organism to which the
construct is to be administered or from another eukaryotic
organism.
[0023] In the nucleic acid constructs the nucleic acid sequence
encoding the mycobacterial stress protein or proline-rich antigen
or antigenic fragment thereof is operably linked to a promoter
capable of expressing the sequence. "Operably linked" refers to a
juxtaposition wherein the promoter and the nucleic acid sequence
encoding the mycobacterial stress protein or proline-rich antigen
or fragment thereof are in a relationship permitting the coding
sequence to be expressed under the control of the promoter. Thus,
there may be elements such as 5' non-coding sequence between the
promoter and coding sequence. These elements may be native either
to the organism from which the promoter sequence is derived or to
the organism from which the mycobacterial stress protein or
proline-rich antigen or fragment thereof is derived. Alternatively,
the said element or elements may be native to neither the organism
from which the promoter sequence is derived nor the organism from
which the mycobacterial stress protein or proline-rich antigen or
fragment thereof is derived. Such sequences can be included in the
construct if they enhance or do not impair the correct control of
the coding sequence by the promoter.
[0024] The expression vector may be of any type. For example, the
vector may be in linear or circular form. It is preferred that the
construct is incorporated into a plasmid vector, since it has been
found that covalent closed circle (CCC) plasmid DNA can be taken up
directly by muscle cells but that the DNA does not integrate into
the genomic DNA of the cells (Ascadi et al, (1991): The New
Biologist; 3, 71-81). Those of skill in the art will be able to
prepare suitable vectors comprising nucleic acid sequences encoding
mycobacterial stress proteins or proline-rich antigens or fragments
thereof starting with widely available vectors which will be
modified by genetic engineering techniques such as those described
by Sambrook et al, (Molecular Cloning: A Laboratory Manual, 1989).
Two suitable starting vectors are the plasmids pCDM8 (Invitrogen;
Seed and Aruffo, Proc. Natl. Acad. Sci. USA (1987)84, 3365-3369)
and pHMG (Gautier et al, Nucl. Acids Res. (1989) 17, 8389).
[0025] Any promoter capable of directing expression of the sequence
encoding the mycobacterial stress protein or proline-rich antigen
or fragment thereof may be operably linked to that sequence.
Particularly suitable promoters are those that direct expression in
a mammalian cell. For example, promoters from viral genes that are
expressed in mammalian cells such as the cytomegalovirus (CMV)
immediate early gene promoter are suitable. Also suitable are
promoters from mammalian genes that are expressed in many or all
mammalian cell types such as the promoters of housekeeping genes.
For example, the p-hydroxymethyl-CoA-reductase (HMG) promoter
(Gautier et al (1989)) is particularly suitable. Also suitable are
promoters and other regulatory elements of genes selectively
expressed in antigen-presenting mammalian cells such as macrophages
and dendritic cells.
[0026] The nucleic acid constructs are useful for gene therapy. In
particular, they are useful for naked DNA vaccination of mammalian
hosts against mycobacterial infections such as those caused by
Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium
bovis. Accordingly, constructs comprising nucleic acid from any
mycobacterial species may be prepared. Owing to the degree of
conservation of some mycobacterial stress proteins, it is not
always necessary to use a nucleic acid sequence from a particular
species to vaccinate against infection by that species. For
example, live BCG (Vacille Calmette-Guerin) cells of Mycobacterium
bovis have long been used to vaccinate humans against Mycobacterium
tuberculosis.
[0027] In the present invention, therefore, a nucleic acid
construct encoding a stress protein or proline-rich antigen of
Mycobacterium tuberculosis, Mycobacterium leprae or Mycobacterium
bovis may be used to vaccinate against infection by any of these
three species of Mycobacterium. For example, the present inventors
have shown that constructs encoding the Mycobacterium leprae 65 kDa
hsp or Mycobacterium leprae 36 kDa proline-rich antigen act as
effective vaccines against Mycobacterium tuberculosis in mice.
[0028] A range of mammalian species can be vaccinated against
mycobacterial infection using the nucleic acid constructs of the
present invention. However, vaccination of humans against
Mycobacterium tuberculosis is particularly desirable. Also
desirable is the vaccination of cattle or deer against
Mycobacterium bovis. Also desirable is the vaccination of badgers
against Mycobacterium bovis as badgers can transmit the bacteria to
cattle.
[0029] The naked nucleic acid constructs of the invention may be
administered to mammals including humans by any route appropriate.
Suitable routes include oral and parenteral, including
subcutaneous, intramuscular, intravenous and intradermal
routes.
[0030] Preferred routes of administration are oral delivery and
injection, typically intramuscular or intradermal injection.
Injection of the vaccine composition into the skeletal muscle or
the skin of the human or animal subject is particularly preferred.
Another mode of delivery of a vaccine composition according to the
invention is by a biolistic or "particle gun" method.
[0031] The naked nucleic acid constructs of the invention may be
administered to the subject alone or in a liposome or associated
with other delivery molecules. The effective dosage depends on many
factors such as whether a delivery molecule is used, the route of
delivery and the size of the mammal being vaccinated. Typical doses
are from 0.1-1000 .mu.g of the nucleic acid construct per dose, for
example 1-500 .mu.g, 50-500 .mu.g, such as 50-75 .mu.g, and 100-500
.mu.g per dose.
[0032] Dosage schedules will vary according to, for example, the
route of administration, the species of the recipient and the
condition of the recipient. However, single doses and multiple
doses spread over periods of days, weeks or months are envisaged.
Single doses typically comprise 0.1-1000 .mu.g, for example 100-500
.mu.g, of nucleic acid and multiple doses typically comprise
0.1-1000 .mu.g, for example 100-500 .mu.g, of nucleic acid each,
preferably in a form suitable for intramuscular or intradermal
injection. Also, single or multiple nucleic acid pellets comprising
a construct according to the invention, for example pellets
comprising 100-500 .mu.g of DNA can be implanted into the recipient
intramuscularly or intradermally. If the construct is administered
by a biolistic method, doses will generally be at the lower end of
the above mentioned ranges, owing the hight efficiency of this
route. Such doses may comprise, for example, 0.1-10 .mu.g, such as
0.1-1 .mu.g, of the construct.
[0033] While it is possible for the naked nucleic acid constructs
of the invention to be administered alone it is preferable to
present them as pharmaceutical formulations. The formulations of
the present invention comprise at least one active ingredient, a
nucleic acid construct according to the invention, together with
one or more acceptable carriers thereof and optionally other
therapeutic ingredients. The carrier or carriers must be
"acceptable" in the sense of being compatible with the other
ingredients of the formulation and not deleterious to the
recipients thereof. Liposomes may be used. Suitable liposomes
include, for example, those comprising the positively charged lipid
(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA), those
comprising dioleoylphosphatidylethanolamine (DOPE), and those
comprising
3.beta.-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol
(Dc-Chol).
[0034] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats, bactericidal
antibiotics and solutes which render the formulation isotonic with
the blood of the intended recipient; and aqueous and non-aqueous
sterile suspensions which may include suspending agents and
thickening agents, and liposomes or other microparticulate systems
which are designed to target the compound to blood components or
one or more organs. The formulations may be presented in unit-dose
or multi-dose containers, for example sealed ampoules and vials,
and may be stored in a freeze-dried (lyophilized) condition
requiring only the addition of the sterile liquid carrier, for
example water, for injections, immediately prior to use. Injection
solutions and suspensions may be prepared extemporaneously from
sterile powders, granules and tablets of the kind previously
described.
[0035] It should be understood that in addition to the ingredients
particularly mentioned above the formulations of this invention may
include other agents conventional in the art having regard to the
type of formulation in question. Of the possible formulations,
sterile pyrogen-free aqueous and non-aqueous solutions are
preferred. Also preferred are formulations in which the nucleic
acid constructs of the invention are contained in liposomes.
[0036] Effective vaccines against mycobacterial infection by
Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium
bovis can also be prepared from bone marrow cells transfected with
a nucleic acid construct which encodes a mycobacterial stress
protein or proline-rich antigen or fragment thereof operably linked
to a promoter capable of directing expression of the said coding
sequence in the said bone marrow cell.
[0037] The bone marrow cells may be transfected by any suitable
method. For example the nucleic acid constructs may be packaged
into infectious viral particles, for example retroviral particles.
This may be done using the methodology described by Silva et al
(1992): Microb. Pathogen. 12, 27-38). The constructs may also be
introduced by electroporation, calcium phosphate precipitation,
biolistic methods or by contacting naked nucleic acid constructs
with the bone marrow cells in solution.
[0038] In the said nucleic acid constructs with which the bone
marrow cells are infected, the nucleic acid may be DNA or RNA,
preferably DNA.
[0039] The nucleic acid constructs with which the bone marrow cells
are transfected may be of any suitable type. Typically, the
constructs will be in the form of an expression vector, such as a
retrovival vector or a naked DNA expression vector as defined
herein. For example, the construct may be in the form of retrovival
shuttle vector derived from the widely available pZipNeo vector or
from a plasmid vector as defined herein, for example pCDM8 or
pHMG.
[0040] The constructs with which the bone marrow cells are
transfected may comprise a coding sequence encoding at least one
further mycobacterial protein or fragment thereof operably linked
to a promoter capable of directing expression of the coding
sequence in the mammalian cell. Typically, the thus encoded further
mycobacterial protein or fragment thereof will be an antigenic
protein or an antigenic fragment thereof. The further mycobacterial
protein or fragment thereof may be a further mycobacterial stress
protein or proline-rich antigen or antigenic fragment thereof.
[0041] The constructs with which the bone marrow cells are
transfected may include any suitable promoter. Particularly
suitable promoters are those that direct expression in a mammalian
cell. For example, promoters from viral genes that are expressed in
mammalian cells such as the cytomegalovirus (CMV) immediate early
gene promoter are suitable. Also suitable are promoters from
mammalian genes that are expressed in many or all mammalian cell
types such as the promoters of housekeeping genes. For example, the
p-hydroxymethyl-CoA-reductase (HMG) promoter (Gautier et al (1989))
is particularly suitable.
[0042] Bone marrow cells transfected with the said constructs may
be administered by any suitable method, such as parenteral
injection, preferably intravenous injection.
[0043] Any effective amount of bone marrow cells transfected with
the said nucleic acid constructs may be administered to the
recipient. Typically, from about 1.times.10.sup.4 to about
1.times.10.sup.8 bone marrow cells are administered, for example
about from 10.sup.5 to 10.sup.7, for example 1.times.10.sup.6 bone
marrow cells.
[0044] The transfected bone marrow cells administered to the
recipient may be of any type that is compatible with the
recipient's immune system. Typically, as for any transplantation of
cells or tissue, the major tissue transplantation antigens of the
administered bone marrow cells will match the major tissue
transplantation antigens of the recipient's cells. The administered
bone marrow cells may be derived from the recipient individual.
[0045] The said transfected bone marrow cells may be delivered to
the recipient alone or in any suitable formulation. A preferred
formulation is a solution that is isotonic with the blood of the
recipient.
[0046] The following Examples illustrate the invention. In the
accompanying drawings:
[0047] FIG. 1 shows the results of DNA Southern blot hybridisation
of DNA extracted from spleen cells of Balb/c mice injected with a
mixture of bone marrow cells infected with retrovirus containing
MLhsp65 nucleic acid and normal bone marrow cells (1:2 ratio);
[0048] FIG. 2 shows the results of Western blot probing of protein
extracted from blood samples from Balb/c mice injected with a
mixture of bone marrow cells infected with retrovirus containing
MLhsp65 nucleic acid and normal bone marrow cells (1:2 ratio);
[0049] FIG. 3 shows the results of a delayed-type hypersensitivity
(DTH) test on mice which had been injected with bone marrow cells
infected with vector that did not contain the MLhsp65 gene
(BMC-Neo), with bone marrow cells infected with retrovirus
containing MLhsp65 nucleic acid (BMC-65) and with recombinant
MLhsp65 (rhsp65);
[0050] FIG. 4 shows the results of challenging with a virulent
strain of M. tuberculosis the groups of mice noted in connection
with FIG. 3 and an additional group immunised with rMLhsp65;
[0051] FIG. 5 depicts plasmid maps of pCDM8ML65 and pHMGML65;
[0052] FIG. 6 shows the number of viable M. tuberculosis in livers
of Balb/c mice injected with intramuscular saline (saline),
rMLhsp65 and Incomplete Freund's Adjuvant (hsp65 IFA), pCDM8ML65
and pHMGML65 (65 DNA), pCDM8 and pHMG each containing the M. leprae
36 kD proline rich antigen gene (36 DNA) and M. bovis BCG;
[0053] FIG. 7 shows the number of viable M. tuberculosis in the
spleen (Sp), liver (Li) and lungs (Lu) of outbred Parkes albino
mice injected with pCDM8 and pHMG (control plasmid DNA), M. bovis
BCG, pCDM8 and pHMG each containing the M. leprae 36 kD proline
rich antigen gene (36 kD plasmid DNA) and pCDM8ML65 and pHMGML65
(65 kD plasmid DNA);
[0054] FIG. 8 shows the number of viable M. tuberculosis in the
spleen (Sp), liver (Li) and lungs (Lu) of CBA/BlO mice injected
with pCDMB and pHMG (control plasmid DNA), M. bovis BCG, pCDM8 and
pHMG each containing the M. leprae 36 kD proline rich antigen gene
(36 kD plasmid DNA) and pCDM8ML65 and pHMGML65 (65 kD plasmid
DNA);
[0055] FIG. 9 shows the number of viable M. tuberculosis in the
spleen (Sp), liver (Li) and lungs (Lu) of untreated Balb/c mice
(naive) or of Balb/c mice injected intradermally with pCDM8ML65
(65id), intramuscularly with pCDM8ML65 (65im), intradermally with
M. bovis BCG or intradermally with pCDM8 (control plasmid)
EXAMPLE 1
[0056] Protection against tuberculosis by the Mycobacterium leprae
hsp65 (MLhsp65) gene expressed in bone marrow cells Bone marrow
cells were removed from the femurs and tibias of Balb/c mice. These
cells were cultured in vitro together with pZIPhsp65 DNA which had
been packaged into infectious retrovirus particles in psi-CRE cells
(Silva et al (1992): Microb. Pathogen. 12, 27-38). pZIPhsp65,
alternatively termed pZIPML65, contains the MLhsp65 gene and is
described in Silva et al (1992). To obtain high efficiencies of
infection and stable integration of viral DNA, the donor mice were
first treated with 5-fluorouracil. High titres of virus
(5.times.10.sup.6 /ml) and polybrene (4 .mu.g/ml), which promotes
viral infection of the cells, were used in vitro. Infected cells
were then selected by including neomycin in the culture medium (0.5
mg/ml) for 2 days.
[0057] Recipient mice were Balb/c, aged 7-8 weeks, and were
.gamma.-irradiated (9.5 Gy) to destroy their bone marrow. They were
immediately injected intravenously with 1.times.10.sup.6 bone
marrow cells. These were either a mixture of cells infected with
the virus containing MLhsp65 nucleic acid, (BMC-65 cells) and
normal bone marrow cells (1:2 ratio) or normal cells alone or cells
that had been infected with the vector without the mycobacterial
gene (BMC-Neo).
[0058] Expression of the mycobacterial gene in recipient animals
was tested 15 days after transplantation by extracting DNA and
protein from spleen cells and blood samples respectively. FIG. 1
shows that, by DNA Southern blot hybridization, spleen cells from
16 to 20 mice contained the MLhsp65 gene. Extracted DNA was blotted
onto nitrocellulose and probed by hydridisation with a M. leprae
3.6 Kb EcoRI DNA fragment containing the MLhsp65 gene (Silva et al
(1992)).
[0059] For a separate group of mice, Western blot probing with a
MLhsp65-specific monoclonal antibody (CL44) revealed that 14 out of
20 had the MLhsp65 protein in their peripheral blood cells (FIG.
2). Recombinant DNA-derived protein (rMLhsp65, 20 ng) and protein
extracted from blood cells of normal mice (cell lysate) were used
as positive and negative controls. Bound antibody was detected with
alkaline phosphatase-conjugated goat anti-mouse Ig.
[0060] 30 days after transplantation of bone marrow, other mice
were tested for delayed-type hypersensitivity (DTH; foot-pad
swelling 48 h after injection of 5 .mu.g rMLhsp65. The results are
shown in FIG. 3. All of 5 mice receiving rMLhsp65 showed DTH
responses, 4 of 9 receiving BMC-65 responded and none of 9
receiving BMC-Neo did.
[0061] 36 days after transplantation they were challenged by
intravenous infection with 5.times.10.sup.6 viable Mycobacterium
tuberculosis H37Rv, a well-known virulent challenge strain obtained
from ATCC, US. 3 weeks after that they were killed and the numbers
of live bacteria in the liver was counted as colony-forming units
on 7H11 agar. An additional group of control mice were immunized
with rMLhsp65 in Freund's incomplete adjuvant (IFA; 25 .mu.g) on
days zero and 7, then boosted on day 14 (15 .mu.g without
adjuvant).
[0062] FIG. 4 shows that the mice that had DTH response after
BMC-65 implantation were also resistant to infection with
Mycobacterium tuberculosis whereas those not responding were not. A
smaller amount of protection was seen in mice responding to
rMLhsp65.
EXAMPLE 2
Protection Against Tuberculosis by Direct Injection of Naked
Mycobacterium leprae hsp65 DNA and Mycobacterium leprae 36 kDa
Proline-Rich Antigen DNA into Muscle
[0063] The gene encoding MLhsp65 was cloned using standard
techniques into two eukaryotic expression vectors pCDM8
(Invitrogen; Seed and Aruffo, Proc. Natl. Acad. Sci. USA (1987)84,
3365-3369) and pHMG (Gautier et al, Nucl. Acids Res. (1989) 17,
8389) to form pCDM8ML65 and pHMGML65 (FIG. 5). Thus, a DNA sequence
comprising the MLhsp65 gene was excised from the E. coli cloning
vector pUC8 using restriction endonucleases (Silva et al (1992)).
This sequence was then ligated into pCDM8 and pHMG. pCDM8 and pHMG
are expression vectors which are not dependent on integration into
the host cell genome and carry strong promoters that are likely to
function in a wide range of mouse cell types.
[0064] The constructs were purified from bulk preparations grown in
E. coli by standard procedures. Normal Balb/c, CBA/BlO or outbred
Parkes albino mice were injected with 50-75 .mu.g of one construct
into the left quadriceps muscle and 50-75 .mu.g of the other
construct into the right quadriceps muscle. The injections were
repeated at intervals of 2-6 weeks until 4 or 5 pairs of injections
had been given in a 3 to 4 month period. 2 weeks after the last
injections the mice were challenged by intraperitoneal infection
with 1.times.10.sup.6 viable Mycobacterium tuberculosis. 6 weeks
after that they were killed and the number of live bacteria in
internal organs was counted as colony-forming units on 7H11
agar.
[0065] Another group of the mice was similarly injected with naked
DNA composed of vectors containing, instead of MLhsp65, the
Mycobacterium leprae gene for a 36 kD proline-rich antigen (Thole
et al, Infection and Immunity (1990) 58, 80-87).
[0066] Two naked DNA constructs containing the 36 kD proline-rich
antigen were in fact prepared. A 1 kb EcoRI fragment was excised
from pTHL1007 (Thole et al (1990)). This fragment was cloned into
the EcoRI polylinker of a cloning vector, pSL301 (Invitrogen;
Brosius, DNA 8, 759-777, 1989; Brosius, Methods in Enzymology 216,
469-483, 1992).
[0067] A 0.9 kb BamHI fragment was excised from the resulting
construct and cloned into the BamHI site of pcDNA1/Neo (Invitrogen;
Wang et al, Cell 67, 797-805, 1991; Spies and DeMars, Nature 351,
323-324, 1991; Seykora et al, Proc. Natl. Acad. Sci. USA 88,
2505-2509, 1991; Attaya et al, Nature 355, 647-648, 1992). The
resulting construct was one of the two constructs containing the
gene for the 36 kD proline-rich antigen of Mycobacterium leprae
that was injected into the mice. The other construct was obtained
again by excising the 0.9 kb BamHI fragment and then cloning it
into the BamHI site of pHMG.
[0068] Finally, further groups were injected with:
[0069] saline only intramuscularly,
[0070] rMLhsp65 protein in IFA as in Example 1,
[0071] Mycobacterium bovis BCG (1.times.10.sup.6 cells,
intradermally on day zero), or
[0072] empty vectors that did not contain inserted genes,
intramuscularly.
[0073] The results are shown in FIGS. 6 to 8. FIG. 6 shows that
Balb/c mice were significantly protected by BCG or by MLhsp65 DNA
(65DNA). The number of live bacteria in the liver of the Balb/c
mice was counted in the case of the results displayed in FIG. 6.
FIGS. 7 and 8 show that both CBA/B10 and the Parkes albino mice
were substantially protected by BCG or by DNA containing either the
MLhsp65 gene or the 36 kD proline-rich antigen gene but not by
empty vectors (control plasmid DNA).
EXAMPLE 3
Protection Against Tuberculosis by Direct Injection of Naked hsp65
DNA into Skin
[0074] pCDM8ML65 DNA was prepared as described above. Balb/c mice
were injected with 50-75 .mu.g of the naked DNA intradermally into
the base of the tail three times at 3 week intervals. Other groups
of the mice were injected at the same times with 100-150 .mu.g of
the same DNA intramuscularly or with 50-75 .mu.g of pCDM8 (vector
only) intradermally. Additional control groups of the mice received
live BCG intradermally as above or were untreated (naive). 8 weeks
after the last DNA injections the mice were challenged by
intraperitoneal infection with 1.times.10.sup.6 viable
Mycobacterium tuberculosis H37Rv. 4 weeks after that they were
killed and the number of live bacteria in the internal organs was
counted as colony-forming units on 7H11 agar. The results are shown
in FIG. 9. The Figure illustrates that the intradermal route gave
effective protection by pCDM8ML65 DNA.
* * * * *